Imidated biopolymer adhesive and hydrogel

ABSTRACT

Biologically compatible polymers carry an imide and can be used as an adhesive, a hydrogel or both. A second biologically compatible polymer reactive with the imidated polymer can be used therewith to seal openings.

BACKGROUND OF INVENTION

Naturally derived biopolymers do not always have the structural orfunctional characteristics required for biomedical applications.Nevertheless, polymeric biomaterials are used in biomedical applicationsincluding medical device coatings, artificial implants, and drugdelivery devices. Polymer networks may be formed, for example, bycrosslinking water soluble polymer solutions to form a wafer insolublepolymer network. Mechanical and structural properties may be manipulatedby modification of the crosslinking density which controls network poresize, water content, and mechanical properties.

Polymers, matrices or gels are attractive for tissue engineering becausethose materials can encapsulate cells. Some polymers or gels have ahigh, tissue-like water content enabling nutrient and waste transport.

SUMMARY OF THE INVENTION

In part, the present disclosure provides for a composition comprising atleast one monomeric unit of a biologically compatible polymerfunctionalized with an imide to provide a tissue adhesive, a hydrogel orboth.

In another embodiment, at least one of the monomeric units of thebiologically compatible polymer is conjugated to a second functionalgroup, which can be an imide. The second functional group, if not animide, can be any known functional group and can provide directionalityto the polymer.

A monomer can be functionalized with at least two functional groups.Overall, when the polymer contains plural species of functional groups,the polymer can contain substantially equal molar amounts of thedifferent functional groups, or the ratios can be varied as a designchoice.

Further, the functionalized biologically compatible polymer compositionsmay comprise at least a second biocompatible polymer that reacts withthe first imidated biological polymer. Thus, the second polymer cancontain functional groups reactive with an imide or another functionalgroup on the first imidated polymer. The functional group on the secondpolymer can be, for example, an amine group.

Compositions of the present disclosure may further comprise abiologically active agent, such as a nutrient a cell, such as a bloodcell or a chondrocyte, or an undifferentiated cell, such as a stem cell,such as a hematopoietic stem cell or a mesenchymal stem cell.

In some embodiments, the compositions of interest are hydrogels withadhesive properties.

The instant invention provides a composition comprising a biologicallycompatible first polymer functionalized with an imide group, andoptionally, a bridging molecule, such as a functionalized secondpolymer, to provide a medical adhesive. In some embodiments, the firstpolymer comprises at least 10 monomeric units, at least 100 monomericunits or at least 1000 or more units of monomer. The bridging moleculecan contain plural functional groups to ensure reaction with at leasttwo molecules of the first polymer.

In a polymer, not all monomers need be functionalized with a reactivemoiety.

The first polymer can be reactive with a surface of a structure, such asa biological structure, such as an organ, tissue or cell, such as acartilage or bone surface, or an artificial structure, such as aprosthesis. A second functional moiety on the first imidated polymer,which may be an imide, also can be reactive with a surface. The firstpolymer can be reactive with a bridging molecule. The reactions can bethrough any means that provide a level of adhesion, such as a covalentbond, a physical crosslinking, an ionic crosslinking or other molecularmechanism that affixes the molecules onto the surface, structure orentity reactive therewith, and with the bridging molecule.

In certain embodiments, multiple polymers are reacted together to form amulti-layer polymer structure with exposed surfaces reactive with asurface, such as a tissue and with the bridging molecule. The bridgingmolecule also can be a multiple layered structure.

Additional features and advantages of the present invention aredescribed in, and will be apparent from the following DetailedDescription of the Invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-20 depict various imides that can be used as reactants toderivatize a monomer or polymer of interest.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention relates in part to a method for filling orfinishing a defect in a tissue or organ, such as sealing an incision orwound. The method comprises applying to the surface a biologicallycompatible polymer comprising an imide. Optionally, the surface canfirst be treated to provide reactive functional groups that can reactwith a first imidated polymer of interest.

Gels, networks, scaffolds, films and the like of interest made with thecomposition(s) of interest encourage cell, tissue and organ integrationand growth. The optional presence of cells, such as stem cells, enhancescell, tissue and organ integration and growth.

Significant to a product of interest is the enhanced integration withthe surrounding tissue to increase stability and bonding to a biologicalsurface and to formation of new tissue. In vitro studies have provenefficacy of the chemical mechanism of reacting to the surface and theincreased mechanical strength of the material-cell/tissue/organinterface.

The instant invention addresses the problem of fibrocartilage formationin a surgical method. The instant invention is usable, for example, inearly osteoarthritic joints by using patches and gels to preventenzymatic synovial degradation during and after implantation. Theinstant invention also enables marrow stimulation without disruptingsubchondral bone integrity. The compound or compounds of interest can beused, for example, in the eye, in the spine, in the musculo-skeletalsystem, at sites carrying cartilage and so on.

The instant invention provides for in situ polymerization techniques toform scaffolds and so on that can be molded to take the desired shape ofthe defect, promote tissue development by stimulating native cellrepair, and can be potentially implanted by minimally invasiveinjection.

This disclosure is directed, at least in part, to polymers, matrices,and gels, and methods of making and using matrices, polymers and gels.One of said such polymers comprises an imide.

For example, this disclosure provides for functionalized biologicallycompatible first polymer, such as hyaluronate, keratan sulfate,chondroitin sulfate and the like, substituted with an imide. SeeWO2006089119, WO2004029137, WO200610516 and WO2006036681, for example,herein incorporated by reference in entirety, for various uses ofsimilar but unrelated compounds. Either imides are not taught therein orthere is an explicit teaching away from the use of imides.

A biological surface refers to an external, environmentally exposedportion of a biological material or entity, such as a microbe, virus,cell, tissue, organ and the like to which a biologically compatiblepolymer can interact react and/or adhere thereto.

A biologically compatible polymer refers to a polymer which isFunctionalized to serve as a composition for applying to a surface. Thepolymer is one that is a naturally occurring polymer or one that is nottoxic to the host. The polymer contains at least an imide. The polymermay be a homopolymer where all monomers are the same or a heteropolymercontaining two or more kinds of monomers. The terms “biocompatiblepolymer”, “biocompatible cross-linked polymer matrix” and“biocompatibility” when used in relation to the instant polymers areart-recognized and are considered equivalent to one another, including,to biologically compatible polymer. For example, biocompatible polymersinclude polymers that are neither toxic to the host (e.g., an animal orhuman), nor degrade (if the polymer degrades) at a rate that producesmonomeric or oligomeric subunits or other byproducts at toxicconcentrations in the host.

An active agent and a biologically active agent are used interchangeablyherein to refer to a chemical or biological compound that induces adesired pharmacological and/or physiological effect, wherein the effectmay be prophylactic or therapeutic. The terms also encompasspharmaceutically acceptable, pharmacologically active derivatives ofthose active agents specifically mentioned herein, including, but notlimited to, salts, esters, amides, prodrugs, active metabolites, analogsand the like. When the terms “active agent,” “pharmacologically activeagent” and “drug” are used, then, it is to be understood that theinvention includes the active agent per se as well as pharmaceuticallyacceptable, pharmacologically active salts, esters, amides, prodrugs,metabolites, analogs etc. The active agent can be a biological entity,such as a virus or cell, whether naturally occurring or manipulated,such as transformed.

Biocompatible polymer, biocompatible cross-linked polymer matrix andbiocompatibility are art-recognized. For example, biocompatible polymersinclude polymers that are neither themselves toxic to the host (e.g., ananimal or human), nor degrade (if the polymer degrades) at a rate thatproduces monomeric or oligomeric subunits or other byproducts at toxicconcentrations in the host. In certain embodiments of the presentinvention, biodegradation generally involves degradation of the polymerin an organism, e.g., into its monomeric subunits, which may be known,to be effectively non-toxic. Intermediate oligomeric products resultingfrom such degradation may have different toxicological properties,however, or biodegradation may involve oxidation or other biochemicalreactions that generate molecules other than monomeric subunits of thepolymer. Consequently, in certain embodiments, toxicology of abiodegradable polymer intended for in vivo use, such as implantation orinjection into a patient, may be determined after one or more toxicityanalyses. It is not necessary that any subject composition have a purityof 100% to be deemed biocompatible; indeed, it is only necessary thatthe subject compositions be biocompatible as set forth above. Hence, asubject composition may comprise polymers comprising 99%, 98%, 97%, 96%,95%, 90%, 85%, 80%, 75% or even less of biocompatible polymers, e.g.,including polymers and other materials and excipients described herein,and still be biocompatible.

To determine whether a polymer or other material is biocompatible, itmay be necessary to conduct a toxicity analysis. Such assays are wellknown in the art. One example of such, an assay may be performed withlive cells, such as HeLa, 293, CHO and the like. The sample is partiallyor completely degraded as known in the art, using, for example, chemicalmeans or enzymatic means. An aliquot of the treated sample products areplaced in culture plates previously seeded with the cells. The sampleproducts are incubated with the cells. The results of the assay may beplotted as % relative growth vs. concentration of degraded sample.

In addition, monomers, polymers, polymer matrices, and formulations ofthe present invention may also be evaluated by well-known in vivo tests,such as subcutaneous implantations in rats to confirm that they do notcause significant levels of irritation or inflammation at thesubcutaneous implantation sites.

Biodegradable is art-recognized, and includes monomers, polymers,polymer matrices, gels, compositions and formulations, such as thosedescribed herein, that are intended to degrade during use, such as invivo. Biodegradable polymers and matrices typically differ fromnon-biodegradable polymers in that the former may be degraded duringuse. In certain embodiments, such use involves in vivo use, such as invivo therapy, and in other certain embodiments, such use involves invitro use. In general, degradation attributable to biodegradabilityinvolves the degradation of a biodegradable polymer into its componentsubunits, or digestion, e.g., by a biochemical process, of the polymerinto smaller, non-polymeric subunits. in certain embodiments, twodifferent types of biodegradation may generally be identified. Forexample, one type of biodegradation may involve cleavage of bonds(whether covalent or otherwise) in the polymer backbone. In suchbiodegradation, monomers and oligomers typically result, and even moretypically, such biodegradation occurs by cleavage of a bond connectingone or more of subunits of a polymer in contrast another type ofbiodegradation may involve cleavage of a bond (whether covalent orotherwise) internal to a side chain or that connects a side chain,functional group and so on to the polymer backbone. For example, atherapeutic agent, biologically active agent, or other chemical moietyattached as a side chain to the polymer backbone may be released bybiodegradation. In certain embodiments, one or the other or both generaltypes of biodegradation may occur during use of a polymer. As usedherein, the term “biodegradation” encompasses both general types ofbiodegradation.

The degradation rate of a biodegradable polymer often depends in part ona variety of factors. Including the chemical identity of the linkageresponsible for any degradation, the molecular weight, crystallinity,biostability, and degree of cross-linking of such polymer, the physicalcharacteristics of the implant, shape and size, and the mode andlocation of administration. For example, the greater the molecularweight, the higher the degree of crystallinity, and/or the greater thebiostability, the biodegradation of any biodegradable polymer is usuallyslower. The term “biodegradable” is intended to cover materials andprocesses also termed “bioerodible”.

In certain embodiments, the biodegradation rate of such polymer may becharacterized by the presence of enzymes, for example, a chondroitinase.In such circumstances, the biodegradation rate may depend on not onlythe chemical identity and physical characteristics of the polymermatrix, but also on the identity of any such enzyme.

In certain embodiments, polymeric formulations of the present inventionbiodegrade within a period that is acceptable in the desiredapplication. In certain embodiments, such as in vivo therapy, suchdegradation occurs in a period usually less than about five years, oneyear, six months, three months, one month, fifteen days, five days,three days, or even one day on exposure to a physiological solution witha pH between 6 and 8 having a temperature of between about 25 and 37° C.In other embodiments, the polymer degrades in a period of between aboutone hour and several weeks, depending on the desired application. Insome embodiments, the polymer or polymer matrix may include a detectableagent that is released on degradation.

Cross-linked herein refers to a composition containing intermolecularcross-links and optionally intramolecular cross-links, arising from,generally, the formation of covalent bonds. Covalent bonding between twocross-linkable components may be direct, in which case an atom in onecomponent is directly bound to an atom in the other component, or it maybe indirect, through a linking group. A cross-linked gel or polymermatrix may, in addition to covalent bonds, also include intermolecularand/or intramolecular noncovalent bonds such as hydrogen bonds andelectrostatic (ionic) bonds.

Functionalized refers to a modification of an existing molecular segmentor group to generate or to introduce a new reactive or more reactivegroup (e.g., imide group) that is capable of undergoing reaction withanother functional group (e.g., an amine group) to form a covalent bond.For example, carboxylic acid groups can be functionalized by reactionwith a carbodiimide and an imide reagent using known procedures toprovide a new reactive functional group in the form of an imide groupsubstituting for the hydrogen in the hydroxyl group of the carboxylfunction.

Got refers to a state of matter between liquid and solid, and isgenerally defined as a cross-linked polymer network swollen in a liquidmedium. Typically, a gel is a two-phase colloidal dispersion containingboth solid and liquid, wherein the amount of solid is greater than thatin the two-phase colloidal dispersion referred to as a “sol.” As such, a“gel” has some of the properties of a liquid (i.e., the shape isresilient and deformable) and some of the properties of a solid (i.e.,the shape is discrete enough to maintain three dimensions on atwo-dimensional surface.)

“Gelation time,” also referred to herein as “gel time,” refers to thetime it takes for a composition to become non-flowable under modeststress. This is generally exhibited as reaching a physical state inwhich the elastic modulus, G′, equals or exceeds the viscous modulus,G″, i.e., when tan (delta) becomes 1 (as may be determined usingconventional rheological techniques).

A hydrogel is a water-swellable polymeric matrix that can absorb waterto form elastic gels, wherein “matrices” are three-dimensional networksof macromolecules held together by covalent or noncovalent crosslinks.On placement in an aqueous environment, dry hydrogels swell by theacquisition of liquid therein to the extent allowed by the degree ofcross-linking.

Hydrogels consist of hydrophilic polymers cross-linked to from awater-swollen, insoluble polymer network. Cross-linking can be initialedby many physical or chemical mechanisms. Photopolymerization is a methodto covalently crosslink polymer chains, whereby a photoinitiator andpolymer solution (termed “pre-gel” solution) are exposed to a lightsource specific to the photoinitiator. On activation, the photoinitiatorreacts with specific functional groups in the polymer chains,crosslinking them to form the hydrogel. The reaction is rapid (3-5minutes) and proceeds at room and body temperature. Photoinducedgelation enables spatial and temporal control of scaffold formation,permitting shape manipulation after injection and during gelation invivo. Cells and bioactive factors can be easily incorporated into thehydrogel scaffold by simply mixing with the polymer solution prior tophotogelation.

Alternatively, the reactants can contain complementary reactive groups,such as an imide and an amine, that yield cross-linking without the needof an external initiator.

Hydrogels of interest can be semi-interpenetrating networks that promotecell, tissue and organ repair while discouraging scar formation. Thehydrogels of interest are derivatized to contain an imide to be reactivewith a surface and/or the second polymer of interest. Hydrogels ofinterest also are configured to have a viscosity that will enable thegelled hydrogel to remain affixed on or in the cell, tissue or organ, orsurface. Viscosity can be controlled by the monomers and polymers used,by the level of water trapped in the hydrogel and by incorporatedthickeners, such as biopolymers, such as proteins, lipids, saccharidesand the like. An example of such a thickener is hyaluronic acid orcollagen.

Polymer is used to refer to molecules composed of repeating monomerunits, including homopolymers, block copolymers, heteropolymers, randomcopolymers, graft copolymers and so on. “Polymers” also include linearpolymers as well as branched polymers, with branched polymers includinghighly branched, dendritic, and star polymers.

A monomer is the basic repeating unit in a polymer. A monomer may itselfbe a monomer or may be dimer or oligomer of at least two differentmonomers, and each dimer or oligomer is repeated in a polymer.

A polymerizing initiator refers to any substance that can initiatepolymerization of monomers or macromers by, for example, free radicalgeneration. The polymerizing initiator often is an oxidizing agent.Exemplary polymerizing initiators include those which are activated byexposure to, for example, electromagnetic radiation or heat.

Incorporated, encapsulated and entrapped are art-recognized when used inreference to a therapeutic agent, dye, or other material and a polymericcomposition, such as a composition of the present invention. In certainembodiments, these terms include incorporating, formulating or otherwiseincluding such agent into a composition that allows for sustainedrelease of such agent in the desired application. The terms maycontemplate any manner by which a therapeutic agent or other material isincorporated into a polymer matrix, including, for example, attached toa monomer of such polymer (by covalent or other binding interaction) andhaving such monomer be part of the polymerization to give a polymericformulation, distributed throughout the polymeric matrix, appended tothe surface of the polymeric matrix (by covalent or other bindinginteractions), encapsulated inside the polymeric matrix, etc. The term“co-incorporation” or “co-encapsulation” refers to the incorporation ofa therapeutic agent or other material and at least one other therapeuticagent or other material in a subject composition.

More specifically, the physical form in which any therapeutic agent orother material is encapsulated in polymers may vary with the particularembodiment. For example, a therapeutic agent or other material may befirst encapsulated in a microsphere and then combined with the polymerin such a way that at least a portion of the microsphere structure ismaintained. Alternatively, a therapeutic agent or other material may besufficiently immiscible in the polymer of the invention that it isdispersed as small droplets, rather than being dissolved, in thepolymer. Any form of encapsulation or incorporation is contemplated bythe present invention, in so much as the sustained release of anyencapsulated therapeutic agent or other material determines whether theform of encapsulation is sufficiently acceptable for any particular use.

Treating or treatment is an art-recognized term which includes curing aswell as ameliorating at least one symptom of any condition or disease.Treating includes preventing a disease, disorder or condition fromoccurring in an animal which may be predisposed to the disease, disorderand/or condition but has not yet been diagnosed as having it, inhibitingthe disease, disorder or condition, e.g., impeding its progress, andrelieving the disease, disorder or condition, e.g., causing any level ofregression of the disease, disorder preventing a disease, disorder orcondition from occurring in an animal which may be predisposed to thedisease, disorder and/or condition but has not yet been diagnosed ashaving it; inhibiting the disease, disorder or condition, e.g., impedingits progress; and relieving the disease, disorder or condition, e.g.,causing regression of the disease, disorder and/or condition. Further,treating the disease or condition includes ameliorating at least onesymptom of the particular disease or condition, even if the underlyingpathophysiology is not affected or other symptoms remain at the samelevel.

Pharmaceutically acceptable salts are art-recognized, and includerelatively non-toxic, inorganic and organic acid addition salts ofcompositions of the present invention, including without limitation,therapeutic agents, excipients, other materials and the like. Examplesof pharmaceutically acceptable salts include those derived from mineralacids, such as hydrochloric acid and sulfuric acid, and those derivedfrom organic acids, such as ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, and the like. Examples of suitable inorganicbases for the formation of salts include the hydroxides, carbonates, andbicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium,aluminum, zinc and the like. Salts may also be formed with suitableorganic bases, including those that are non-toxic and strong enough toform such salts. For purposes of illustration, the class of such organicbases may include mono-, di-, and trialkylamines, such as methylamine,dimethylamine, and triethylamine; mono-, di- or trihydroxyalkylaminessuch as mono-, di-, and triethanolamine; amino acids, such as arginineand lysine; guanidine; N-methylglucosamine, N-methylglucamine;L-glutamine; N-methylpiperazine; morpholine; ethylenediamine;N-benzylphenethylamine; (trihydroxymethyl) aminoethane; and the like,see, for example, J. Pharm. Sci., 66: 1-19 (1977).

Prophylactic or therapeutic treatment is art-recognized and includesadministration to the host of one or more of the subject compositions.If it is administered prior to clinical manifestation of the unwantedcondition (e.g., disease or other unwanted state of the host animal)then the treatment is prophylactic, i.e., it protects the host againstdeveloping the unwanted condition, whereas if it is administered aftermanifestation of the unwanted condition, the treatment, is therapeutic(i.e., it is intended to diminish, ameliorate, or stabilize the existingunwanted condition or side effects thereof).

The term “aliphatic” is an art-recognized term, and includes linear,branched, and cyclic alkanes, alkenes or alkynes. In certainembodiments, aliphatic groups in the present invention are linear orbranched and have from 1 to about 20 carbon atoms.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chainalkyl-groups, cycloalkyl (alicyclic) groups, alkyl substitutedcycloalkyl groups, and cycloalkyl substituted alkyl groups. In certainembodiments, a straight chain or branched chain alkyl has about 30 orfewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chain,C₃-C₃₀ for branched chain), and alternatively, about 20 or fewer carbonatoms. Likewise, cycloalkyls have from about 3 to about 10 carbon atomsin their ring structure, and alternatively about 5, 6 or 7 carbons inthe ring structure.

Moreover, the term “alkyl” (or “lower alkyl”) includes both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents mayinclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphonate, a phosphinate, an amino, an amido, anamidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, analkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamide, asulfnoyl, a heterocycyl, an aralkyl or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain may themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylsmay be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN and the like.

The term “aralkyl” is art-recognized, and includes aryl groups (e.g., anaromatic or heteroaromatic group).

The terms “alkenyl” and “alkynyl” are art-recognized, and includeunsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond, respectively.

The term “heteroatom” is art-recognized, and in an organic molecule,generally includes an atom of any element other than carbon or hydrogen.Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus,sulfur and selenium.

The term “aryl” is art-recognized, and includes 5-, 6- and 7-memberedsingle ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiaxole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring may be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls, or rings joined by non-cyclic moieties.

The terms “ortho”, “meta” and “para” are art-recognized and apply to1,2-, 1,3- and 1,4- disubstituted cyclohexanes, respectively. Forexample, the names 1,2-dimethylbenzene and ortho-dimethylbenzene aresynonymous.

The terms “heterocyclyl” and “heterocyclic group” are art-recognized,and include 3- to about 10-membered ring structures, such as 3- to about7-membered rings, whose ring structures include one to four heteroatoms.Heterocycles may also be polycycles. Heterocyclyl groups include, forexample, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene,xanthene, phenoxanthin, pyrrole, imidazole, pyrazole, isothiazole,isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine,isoindole, indole, indazole, purine, quinolizine, isoquinoline,quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline,cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine,pyrimidine, phenanthroline, phenazine, phenarsazine, phenothizine,furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole,piperidine, piperazine, morpholine, lactones, lactams such asazetidinones and pyrrolidinones, sultams, sultones and the like. Theheterocyclic ring may be substituted atone or more positions with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic heteroaromatic moiety, —CF₃, —CN or the like.

The terms “polycyclyl” and “polycyclic group” are art-recognized, andinclude structures with two or more rings (e.g., cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which twoor more carbons are common to two adjoining rings, e.g., the rings are“fused rings”. Rings that are joined through non-adjacent atoms, e.g.,three or more atoms are common to both rings, are termed “bridged”rings. Each of the rings of the polycycle may be substituted with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxylsilyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN or thelike.

The term “carbocycle” is art recognized and includes an aromatic ornon-aromatic ring in which each atom of the ring is carbon. Thefollowing art-recognized terms have the following meanings: “nitro”means —NO₂; the term “halogen” designates —F, —Cl, —Br or —I; the term“sulfhydryl” means —SH; the term “hydroxyl” or “hydroxy” means —OH; andthe term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and include bothunsubstituted and substituted amines. A primary amine carries twohydrogens, a secondary amine, one hydrogen and another substituent and atertiary amine, the two hydrogens are substituted. The substituents forone or both of the hydrogens can be, for example, an alkyl, an alkenyl,an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, a polycycle and soon. If both hydrogens are substituted with carbonyls, the carbonylframed nitrogen forms an imide.

The term “alkylamine” includes an amine group, as defined above, havinga substituted or unsubstituted alkyl attached thereto.

The term “amido” is art-recognized as an amino-substituted carbonyl.

The term “alkylthio” is art-recognized and includes an alkyl group, asdefined above, having a sulfur radical attached thereto. In certainembodiments, the “alkylthio” moiety is represented by one of —S-alkyl,—S-alkenyl, —S-alkynyl and so on. Representative alkylthio groupsinclude methylthio, ethylthio and the like.

The term “carbonyl” is art-recognized and includes a C═O structure.Carbonyls are involved in esters; carboxyl groups; formates;thiocarbonyls; thioesters; thiocarboxylic acids; thioformates; ketones;and aldehydes.

The terms “alkoxyl” and “alkoxy” are art-recognized and include an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like.

An “ether” is two hydrocarbons covalently linked by an oxygen.Accordingly, the substituent of an alkyl that renders that alkyl anether is or resembles an alkoxyl, such as may be represented by one of—O-alkyl, —O-alkenyl, —O-alkynyl and so on.

The term “sulfonate” is art-recognized and includes a moiety wherein asulfur atom carries two double bonded oxygens and a single bondedoxygen.

The term “sulfate” is art-recognized and includes a moiety thatresembles a sulfonate but includes two single bonded oxygens.

The terms “sulfonamide,” “sulfamoyl,” “sulfonyl” and “sulfoxido” areart-recognized and each can include a variety of R group substituents asdescribed herein.

The terms “phosphoramidite” and “phosphonamidite” are art-recognized.

The term “selenoalkyl” is art-recognized and includes an alkyl grouphaving a substituted seleno group attached thereto. Exemplary“selenoethers” which may be substituted on the alkyl are selected fromone of —Se-alkyl, —Se-alkenyl, —Se-alkynyl and so on.

Substitutions may be made to alkenyl and alkynyl groups to produce, forexample, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls,iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

A hydrocarbon is an art recognized term and includes all permissiblecompounds having at least one hydrogen and one carbon atom. For example,permissible hydrocarbons include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticorganic compounds that may be substituted or unsubstituted.

The phrase “protecting group” is art-recognized and includes temporarysubstituents that protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, siyl ethers of alcohols, and acetalsand ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed, Greene et al., ProtectiveGroups in Organic Synthesis 2nd ed., Wiley, New York, (1991), forexample.

The definition of each expression, e.g. alkyl, aryl etc., when it occursmore than once in any structure, is intended to be independent of itsdefinition elsewhere in the same structure unless otherwise indicatedexpressly or by the context.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules, that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms are art-recognized andrepresent methyl, ethyl, phenyl, trifluoromethanesulfonyl,nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl,respectively. A more comprehensive list of the abbreviations utilized byorganic chemists of ordinary skill in the art appears in the first issueof each volume of the Journal of Organic Chemistry; this list istypically presented in a table entitled Standard List of Abbreviations.

Certain monomeric subunits of the present invention may exist inparticular geometric or stereoisomeric forms. In addition, polymers andother compositions of the present invention may also be opticallyactive. The present invention contemplates all such compounds, includingcis-isomers and trans-isomers, R-enantiomers and S-enantiomers,diastereomers, (d)-isomers, (l)-isomers, the racemic mixtures thereof,and other mixtures thereof as falling within the scope of the invention.Additional asymmetric carbon atoms may be present in a substituent suchas an alkyl group. All such isomers, as well as mixtures thereof, areintended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

It will be understood that substitution or substituted with includes theimplicit proviso that such substitution is in accordance with thepermitted valency of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation, such as by rearrangement,cyclization, elimination, or other reaction.

The term substituted is also contemplated to include all permissiblesubstituents of organic compounds such as the imide reagent of interest.In a broad aspect, the permissible substituents include acyclic andcyclic, branched and unbranched, carbocyclic and heterocyclic, aromaticand nonaromatic substituents of organic compounds. Illustrativesubstituents include, for example, those described hereinabove. Thepermissible substituents may be one or more and the same or differentfor appropriate organic compounds. For purposes of this invention, theheteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. This invention is not intendedto be limited in any manner by the permissible substituents of organiccompounds.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87.

A functional group or a moiety which can be used for substitution is onecapable of mediating formation of a polymer or reaction with a surfaceor other molecule. Functional groups include the various radicals andchemical entities taught herein, and include alkenyl moieties such asacrylates, methacrylates, dimethacrylates, oligoacrylates,oligomethacrylates, ethacrylates, itaconates or acrylamides. Furtherfunctional groups include aldehydes. Other functional groups may includeethylenically unsaturated monomers including, for example, alkyl estersof acrylic or methacrylic acid such as methyl methacrylate, ethylmethacrylate, butyl methacrylate, ethyl acrylate, butyl acrylate, hexylacrylate, n-octyl acrylate, lauryl methacrylate, 2-ethylhexylmethacrylate, nonyl acrylate, benzyl methacrylate, the hydroxyalkylesters of the same acids such as 2-hydroxy ethyl acrylate,2-hydroxyethyl methacrylate, and 2-hydroxypropyl methacrylate, thenitrile and amides of the same acids such as acrylonitrile,methyacrylonitrile, and methacrylamide, vinyl acetate, vinyl propionate,vinylidene chloride, vinyl chloride, and vinyl aromatic compounds suchas styrene, t-butyl styrene and vinyl toluene, dialkyl maleates, dialkylitaconates, dialkyl methylene-malonates, isoprene and butadiene.Suitable ethylenically unsaturated monomers containing carboxylic acidgroups include acrylic monomers, such as acrylic acid, methacrylic acid,ethacrylic acid, itaconic acid, maleic acid, fumaric acid, monoalkylitaconate including monomethyl itaconate, monoethyl itaconate, andmonobutyl itaconate, monoalkyl maleate including monomethyl maleate,monoethyl maleate, and monobutyl maleate, citraconic acid and styrenecarboxylic acid. Suitable polyethylenically unsaturated monomers includebutadiene, isoprene, allylmethacrylate, diacrylates of alkyl diols suchas butanediol diacrylate and hexanediol diacrylate, divinyl benzene andthe like.

In some embodiments, a monomeric unit of a biologically compatiblepolymer may be functionalized through one or more thio, carboxylic acidor alcohol moieties located on a monomer of the biopolymer. For example,in the case of chondroitin sulfate, a carbonyl group can be derivatizedwith a imide group using, for example, carbodiimide chemistry. Analcohol group can be derivatized using, for example, the Mitsunobureaction, Procter et al., Tetra. Lett. 47(20) 5151-5154, 2006.

In some embodiments, this disclosure is directed to a compositioncomprising at least one monomeric unit of a biologically compatiblepolymer, such as hyaluronic acid, heparin sulfate, keratan sulfate andthe like, functionalized by an imide. Those starting molecules arenatural components of extracellular matrices. However, in general, anybiologically compatible polymer can be used as the polymer, whichpolymer carries at least an imide. Other suitable polymers include thosewhich are naturally occurring, such as a GAG, mucopolysaccharide,collagen, or proteoglycan components, such as hyaluronic acid, heparinsulfate, glucosamines, dermatans, keratans, heparans, hyalurunan,aggrecan and the like.

In some embodiments, this disclosure is directed to a compositioncomprising at least one monomeric unit of a saccharide or otherbiocompatible monomer or polymer, wherein the monomers have reactivesites that will enable at least inclusion of an imide and otherfunctional groups, such as chondroitin sulfate. Chondroitin sulfate is anatural component of cartilage and may be a useful scaffold material forregeneration. Chondroitin sulfate includes members of 10-60 kDaglycosaminoglycans. The repeat units, or monomeric units, of chondroitinsulfate consist of a disaccharide, β(1→4)-linked D-glucuronyl β(1→3)N-acetyl-D-galactosamine sulfate.

The imide of the instant invention is not limited to any one reactant asmany are known in the art and are usable in the context of the instantinvention, that is, to provide an intermediate derivative that does notspontaneously degrade rapidly but is stable enough to react with, forexample, an amine on a desired molecule, wherein the imide, ifhydroxylated and the oxygen thereof is the bonding site, is regeneratedas a hydroxyimide and replaced by a functional group on the desiredmolecule. Examples of imides are provided in FIGS. 1-20, such assuccinimide.

Cross-linked polymer matrices of the present invention may include andform hydrogels. The water content of a hydrogel may provide informationon the pore structure. Further, the water content may be a factor thatinfluences, for example, the survival of encapsulated cells within thehydrogel. The amount of water that a hydrogel is able to absorb may berelated to the cross-linking density and/or pore size. For example, thepercentage of imides on a functionalized macromer, such as chondroitinsulfate or keratin sulfate, may dictate the amount of water that isabsorbable.

The compositions of the present invention may comprise monomers,macromers, oligomers, polymers, or a mixture thereof. The polymercompositions can consist solely of covalently crosslinkable polymers, orionically crosslinkable polymers, or polymers crosslinkable by redoxchemistry, or polymers crosslinked by hydrogen bonding, or anycombination thereof. The reagents should be substantially hydrophilicand biocompatible.

Suitable hydrophilic polymers to serve as the first and second polymersinclude synthetic polymers such as poly(ethylene glycol), poly(ethyleneoxide), partially or fully hydrolyzed poly(vinyl alcohol),poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethyleneoxide)-co-poly(propylene oxide) block copolymers (poloxamers andmeroxapols), poloxamines, carboxymethyl cellulose, and hydroxy alkylatedcelluloses such as hydroxyethyl cellulose and methylhydroxypropylcellulose, and natural polymers such as polypeptides, polysaccharides orcarbohydrates such as Ficoll™, polysucrose, hyaluronic acid, dextran,heparan, sulfate, chondroitin sulfate, heparin, or alginate, andproteins such as gelatin, collagen, albumin, or ovalbumin or copolymersor blends thereof. As used herein, “celluloses” includes cellulose andderivatives of the types described above; “dextran” includes dextran andsimilar derivatives thereof.

Polysaccharides or other biologically compatible polymers that are veryviscous liquids or that are thixotropic, and form a gel over time by theslow evolution of structure, are also useful. For example, hyaluronicacid, which can form an injectable gel with a consistency like a hairgel, may be utilized. Modified hyaluronic acid derivatives areparticularly useful. As used herein, the term, “modified hyaluronicacids” refers to chemically modified hyaluronic acids. Modifiedhyaluronic acids may be designed and synthesized with preselectedchemical modifications to adjust the rate and degree of crosslinking andbiodegradation. For example, modified hyaluronic acids may be designedand synthesized which are esterified with a relatively hydrophobic groupsuch as propionic acid or benzylic acid to render the polymer morehydrophobic and gel-forming, or which are grafted with amines to promoteelectrostatic self-assembly. Modified hyaluronic acids thus may besynthesized which are injectable, in that they flow under stress, butmaintain a gel-like structure when not under stress. Hyaluronic acid andhyaluronic derivatives are available from Genzyme, Cambridge, Mass. andFidia, Italy.

Alternatively, a biologically compatible polymer can be incorporatedinto a matrix of interest to form a composite. Hence, a molecule, suchas hyaluronic acid or a collagen can be incorporated into a matrix ofinterest. Reactivity of that incorporated biopolymer can be desired.Hence, amine groups on the introduced polymer can be reactive with thematrix components of interest, which may yield a composite structure ofhigher modulus. Alternatively, to gain the benefit of the polymer to thecomposite properties without impacting modulus substantially, such as toretain tissue adhesiveness, the introduced polymer can be functionalizedto reduce activity of any reactive functions thereon. Thus, for example,the amines of a polymer, such as collagen, can be functionalized, forexample, to carry an alkyl group, an acetyl group and so on as taughtherein to yield a polymer less reactive with imide groups.

Methods for the synthesis of the polymers described above are known tothose skilled in the art, see, for example Concise Encyclopedia ofPolymer Science and Polymeric Amines and Ammonium Salts, E. Goethals,editor (Pergamen Press, Elmsford, N.Y. 1980). Many polymers, such aspoly(acrylic acid), are commercially available. Naturally occurringpolymers can be isolated from biological sources as known in the art orare commercially available. Naturally occurring and synthetic polymersmay be modified using chemical reactions available in the art anddescribed, for example, in March, “Advanced Organic Chemistry,” 4thEdition, 1992, Wiley-Interscience Publication, New York.

Representative embodiments of the invention include a method ofproducing an imidated saccharide, monomer or polymer moiety, where themethod can include use of a, for example, carbodiimide intermediate, andan imide reactant to form the imide-derivatized monomer. Examples ofcarbodiimides include N,N′-dicylohexylcarbodimide (DCC),N,N′-diisopropylcarbodimide (DIC) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Othermethods for imidating a molecule are known in the art.

Numerous chemical options are available for modifying polymers that maythen undergo a radical polymerization. For example, methacrylicanhydride, methacryloyl chloride and glycidyl methacrylate may be usedto add methacrylate groups to one or more monomers of a polymer chain.Glycidyl methacrylate may be used, for example, for efficiency ofreaction. Further, the modification reagents may be chosen to optimize alack of cytotoxic byproducts.

In some embodiments, the number of each of the functional groups perpolymeric unit may be at least one moiety per about 10 monomeric units,at least about 2 moieties per about 10 monomeric units up through one ormore functional groups per monomer. Alternatively, the number offunctional groups per polymeric unit may be at least one moiety perabout 12 monomeric units, per about 14 monomeric units of more. Forexample, there may be at least about one imide group per ten monomericunits.

Moreover the ratio of each of the imide and other functional group canbe 5:1, 9:2, 4:1, 7:2, 3:1, 5:2, 2:1, 3:2, 1:1, 2:3, 1:2, 2:5, 1:3, 2:7,1:4, 2:9 or 1:5 in along the full length of the polymer. Preferably,each of the imide and other functional group is regularly distributedalong the length of the polymer. However, the arrangement of thefunctional groups can be configured to be non-random or regularinterposed, for example, to be concentrated at certain sites of thepolymer backbone for an intended use. Hence, the groups can be isolatedat different portions of the polymer. If aside from the imide there aretwo or more other functional groups, the ratio of each of the functionalgroups to one another can vary from unity to any other ratio or ratiosas a design choice.

A composition comprising a cross-linked polymer matrix, wherein saidcross-linked polymer matrix, comprises at least one imidatedbiologically compatible polymer is provided. In some embodiments, saidcross-linked polymer matrix further comprises at least two imidatedbiocompatible polymers. In other embodiments, a cross-linked polymermatrix further comprises a second biocompatible polymer comprising oneor more functional groups reactive with the functional groups on saidfirst imidated polymer, such as amine groups, such as, for example,found on a protein.

The accompanying molecule that can bind the imidated biopolymers ofinterest together, such as a polymer carrying amines, the bridgingmolecule, generally is a polymer that contains plural reactive sites,wherein said reactive sites are those which react with sites found on animidated biologically compatible polymer of interest. The bridgingmolecule preferably is biocompatible. The bridging molecule, as with theimidated biologically compatible polymer, can be biodegradable. Thebridging molecule can be configured into a multiple layered structure,wherein the internal layers can be the same or different so long as thesuperficial, external layers present with exposed functional group forreacting with sites on the imidated polymer which may be atissue-adhered polymer.

Suitable polymers for the imidated polymer and bridging molecule ofinterest include biocompatible monomers with recurring units found inpoly(phosphoesters), poly(lactides), poly(glycolides),poly(caprolactones), poly(anhydrides), poly(amides), poly(urethanes),poly(esteramides), poly(orthoesters), poly(dioxanones), poly(acetals),poly(ketals), poly(carbonates), poly(orthocarbonates),poly(phosphazenes), poly(hydroxybutyates), poly(hydroxyl valerates),poly(alkylene oxalates), poly(alkylene succinates), poly(malic acids),poly(amino acids), polyvinylalcohol, poly(vinylpyrrolidone),poly(ethylene glycol ), poly(hydroxycellulose), chitin, chitosan, andcopolymers, terpolymers or combinations or mixtures of the abovematerials. For example, a polymer can be substituted to carry aminegroups.

Other suitable synthetic polymers include polymers containing aminegroups, such as chemically synthesized polypeptides. Such polypeptidesmay include polynucleophilic polypeptides that have been synthesized toincorporate amino acids containing primary amino groups for example,lysine, and/or amino acids containing thiol groups (such as cysteine).Further suitable synthetic polymers include poly(amino)acids.

Other compounds that may include amine groups include proteins such asalbumin. Albumin may be of mammalian origin, but other sources ofalbumin also may be employed. Bovine serum albumin (BSA) may be used,for example. Alternatively, albumin may be recombinant albumin, isolatedfrom cells expressing a recombinant albumin gene, using methods known inthe art. Major fragments of albumin, comprising at least 100 residues ofan albumin sequence, whether produced by partial proteolysis or byrecombinant means, may also be used instead of intact albumin as abridging molecule. Alternatively, useful fragments may contain at least50 residues, and more preferably at least 75 residues of an albuminsequence. Finally, mixtures of different forms of albumin (e.g., human,bovine, recombinant, fragmented), and plasma fractions rich in albuminmay also be employed. Albumin may be purified directly from tissues orcells, using methods well known in the art.

When used, polymerizing initiators include electromechanical radiation.Initiation of polymerization may be accomplished by irradiation withlight at a wavelength of between about 200 to about 700 nm, or aboveabout 320 nm or higher, or even about 365 nm.

Examples of other initiators are organic solvent-soluble initiators suchas benzoyl peroxide, azobisisobutylronitrile (AIBN), di-tertiary butylperoxide and the like, water soluble initiators such as ammoniumpersulfate (APS), potassium, persulfate, sodium persulfate, sodiumthiosulfate and the like, redox-type initiators which are combinationsof such initiator and tetramethylethylene, Fe²⁺ salt, sodium, hydrogen,sulfite or like reducing agent etc.

Useful photoinitiators are those which can be used to initiate by freeradical generation polymerization of monomers with minimal cytotoxicity.In some embodiments, the initiators may work in a short time frame, forexample, minutes or seconds. Exemplary dyes for UV or visible lightinitiation include ethyl eosin, 2,2-dimethoxy-2-phenyl acetophenone,2-methoxy-2-phenylacetophenone, other acetophenone derivatives, andcamphorquinone.

Other photooxidizable and photoreducible dyes that may be used toinitiate polymerization include acridine dyes, for example, acriblarine;thiazine dyes, for example, thionine; xanthine dyes, for example, rosebengal; and phenazine dyes, for example, methylene blue. These may beused with cocatalysts such, as amines, for example, triethanolamine;sulphur compounds; heterocyclics, for example, imidazole; enolates;organometallics; and other compounds, such as N-phenyl glycine. Otherinitiators include campborquinones and acetophenone derivatives.

Thermal polymerization initiator systems may also be used. Such systemsthat are unstable at 37° C. and would initiate free radicalpolymerization at physiological temperatures include, for example,potassium persulfate, with or without tetramethyl ethylenediamine;benzoylperoxide, with or without triethanolamine; and ammonium,persulfate with sodium bisulfite.

Alternatively, the first imidated polymer may react spontaneously with asurface, such as a tissue or prosthesis. The first imidated polymer alsocan react with the second biocompatible polymer. The two reactants canbe mixed prior to application, applied simultaneously and so on as knownin the art to provide polymerization of the two polymers. An initiatoris used, as needed, as a design choice.

Cross-linked polymer matrices of the present invention may form and mayinclude hydrogels. The water content of a hydrogel may provideinformation on the pore structure. Further, the water content may be afactor that influences, for example, the survival of encapsulated cellswithin the hydrogel. The amount of water that is able to be absorbed maybe related to the cross-linking density pore size. For example, thepercentage of methacrylate groups on a functionalized polymer maydictate the amount of water absorbable.

For example, poly(ethylene oxide)-diacrylate (PEODA) carrying an insidemay be used in a polymer system for tissue engineering, and cross-linkedpolymer matrices may include cogels of CS-I (chondroitin sulfate-imide)and PEODA.

The mechanical properties of a cross-linked polymer matrix, such as ascaffold, may also be related to the pore structure. For applications intissue engineering, scaffolds with different mechanical properties maybe desirable depending on desired clinical application. For example,scaffolds for cartilage tissue engineering in the articular joint mustsurvive higher mechanical stresses than a cartilage tissue engineeringsystem in other body sites. Thus, polymers with mechanical propertiesthat are easily manipulated may be desired, and can be obtained as adesign choice.

Cytotoxicity of the biopolymer scaffold system may be evaluated with anysuitable cells, such as fibroblasts, by, for example, using a live-deadfluorescent cell assay and MTT, a compound that actively metabolizingcells convert from yellow to purple, as taught hereinabove, and as knownin the art.

In one aspect of this invention, a composition comprising a cross-linkedpolymer matrix or gel and one or more biologically active agents may beprepared. The biologically active agent may vary widely with theintended purpose for the composition. The term active is art-recognizedand refers to any moiety that is a biologically, physiologically, orpharmacologically active substance that acts locally or systemically ina subject. Examples of biologically active agents, that may be referredto as “drugs”, are described in well-known literature references such asthe Merck Index, the Physicians Desk Reference, and The PharmacologicalBasis of Therapeutics, and they include, without limitation,medicaments; vitamins; mineral supplements; substances used for thetreatment, prevention, diagnosis, cure or mitigation of a disease orillness; substances which affect the structure or function of the body,or pro-drugs, which become biologically active or more active after theyhave been placed in a physiological environment. Various forms of abiologically active agent may be used which are capable of beingreleased the subject composition, for example, into adjacent tissues orfluids upon administration to a subject. In some embodiments, abiologically active agent may be used in cross-linked polymer matrix ofthis invention, to, for example, promote cartilage formation. In otherembodiments, a biologically active agent may be used in cross-linkedpolymer matrix of this invention, to treat, ameliorate, inhibit, orprevent a disease or symptom, in conjunction with, for example,promoting cartilage formation.

Further examples of biologically active agents include, withoutlimitation, enzymes, receptor antagonists or agonists, hormones, growthmeters, autogenous bone marrow, antibiotics, antimicrobial agents andantibodies. The term “biologically active agent” is also intended toencompass various cell types and genes that can be incorporated into thecompositions of the invention.

In certain embodiments, the subject compositions comprise about 1% toabout 75% or more by weight of the total composition, alternativelyabout 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60% or 70%, of a biologicallyactive agent.

Non-limiting examples of biologically active agents include following:adrenergic blocking agents, anabolic agents, androgenic steroids,antacids, anti-asthmatic agents, anti-allergenic materials,anti-cholesterolemic and anti-lipid agents, anti-cholinergics andsympathomimetics, anti-coagulants, anti-convulsants, anti-diarrheal-,anti-emetics, anti-hypertensive agents, anti-infective agents,anti-inflammatory agents such as steroids, non-steroidalanti-inflammatory agents, anti-malarials, anti-manic agents,anti-nauseants, anti-neoplastic agents, anti-obesity agents,and-parkinsonian agents, anti-pyretic and analgesic agents,anti-spasmodic agents, anti-thrombotic agents, anti-uricemic agents,anti-anginal agents, anti-histamines, anti-tussives, appetitesuppressants, benzophenanthridine alkaloids, biologicals, cardioactiveagents, cerebral dilators, coronary dilators, decongestants, diuretics,diagnostic agents, erythropoietic agents, estrogens, expectorants,gastrointestinal sedatives, agents, hyperglycemic agents, hypnotics,hypoglycemic agents, ion exchange resins, laxatives, mineralsupplements, mitotics, mucolytic agents, growth factors, neuromusculardrugs, nutritional substances, peripheral vasodilators, progestationalagents, prostaglandins, psychic energizers, psychotropics, sedatives,stimulants, thyroid and antithyroid agents, tranquilizers, uterinerelaxants, vitamins, antigenic materials, and prodrugs.

Specific examples of useful biologically active agents the abovecategories include: (a) anti-neoplastics such as androgen, inhibitors,antimetabolites, cytotoxic agents, and immunomodulators; (b)anti-tussives such as dextromethorphan, hydrobromide, noscapine,carbetapentane citrate, and chlophedianol hydrochloride; (c)antihistamines such, as chlorpheniramine phenindamine tartrate,pyrilamine doxylamine succinate, and phenyltoloxamine citrate; (d)decongestants such as hydrochloride, phenylpropanolamine hydrochloride,pseudoephedrine hydrochloride, and ephedrine; (e) various alkaloidssuch, as codeine phosphate, codeine sulfate, and morphine; (f) mineralsupplements such as potassium chloride, zinc chloride, calciumcarbonate, magnesium oxide, and other alkali metal, and alkaline earthmetal salts; (g) ion exchange resins such as such asN-acetylprocainamide; (i) antipyretics and analgesics such asacetaminophen, aspirin and ibuprofen; appetite suppressants such asphenyl-propanolamine or caffeine; (k) expectorants such as guaifenesin;(l) antacids such as aluminum hydroxide and magnesium hydroxide;biologicals such as peptides, polypeptides, proteins and amino acids,hormones, interferons or cytokines and other bioactive peptidecompounds, such as calcitonin, ANF, EPO and insulin; (n) anti-infectiveagents such as anti-fungals, anti-virals, antiseptics and antibiotics;and (m) desensitizing agents and antigenic materials, such as thoseuseful for vaccine applications.

More specifically, non-limiting examples of useful biologically activeagents include the following therapeutic categories: analgesics, such asnonsteroidal anti-inflammatory drugs, opiate agonists and salicylates;antihistamines, such as H1-blockers and H2-blockers; anti-infectiveagents, such as antihelmintics, antiaeorobics, antibiotics,aminoglycoside antibiotics, antifungal antibiotics, cephalosporinantibiotics, macrolide antibiotics, miscellaneous antibiotics,penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics,tetracycline antibiotics, antimycobacterials, antituberculosisantimycobacterials, antiprotozoals, antimalarial antiprotozoals,antiviral agents, anti-retroviral agents, scabicides, and urinaryanti-infectives; antineoplastic agents, such as alkylating agents,nitrogen mustard alkylating agents, nitrosourea, alkylating agents,antimetabolites, purine analog antimetabolites, pyrimidine analogantimetabolites, hormonal antineoplastics, natural antineoplastics,antibiotic natural antineoplastics, and vinca alkaloid naturalantineoplastics; autonomic agents, such as anticholinergics,antimuscarinic anticholinergics, ergot alkaloids, parasympathomimetics,cholinergic agonist parasympathomimetics, cholinesterase inhibitorparasympathomimetics, sympatholytics, α-blocker sympatholytics,sympatholytics, sympathomimetics, and adrenergic agonistsympathomimetics; cardiovascular agents, such as antianginals,antianginals, calcium-channel blocker antianginals, nitrateantianginals, antiarrhythmics, cardiac glycoside antiarrhythmics, classI antiarrhythmics, class antiarrhythmics, class antiarrhythmics, classIV antiarrhythmics, antihypertensive agents, α-blockerantihypertensives, angiotensin-converting enzyme inhibitor (ACEinhibitor) antihypertensives, 13-blocker antihypertensives,calcium-channel blocker antihypertensives, central-acting adrenergicantihypertensives, diuretic antihypertensive agents, peripheralvasodilator antihypertensives, antilipemics, bile acid sequestrantantilipemics, reductase inhibitor antilipemics, inotropes, cardiacglycoside inotropes, and thrombolytic agents; dermatological agents,such as antihistamines, anti-inflammatory agents, corticosteroidanti-inflammatory agents, anesthetics, topical anti-infectives, topicalanti-infectives, antiviral topical anti-infectives, and topicalantineoplastics; electrolytic and renal agents, such as acidifyingagents, alkalinizing agents, diuretics, carbonic anhydrase inhibitordiuretics, loop diuretics, osmotic diuretics, potassium-sparingdiuretics, thiazide diuretics, electrolyte replacements, and uricosuricagents; enzymes, such as pancreatic enzymes and thrombolytic enzymes;gastrointestinal agents, such as antidiarrheals, antiemetics,gastrointestinal anti-inflammatory agents, salicylate gastrointestinalanti-inflammatory agents, antacid anti-ulcer agents, gastric acid-pumpinhibitor anti-ulcer agents, gastric mucosal anti-ulcer agents,H2-blocker anti-ulcer agents, cholelitholytic agents, digestants,emetics, laxatives and stool softeners, and prokinetic agents; generalanesthetics, such as inhalation, anesthetics, halogenated inhalationanesthetics, intravenous anesthetics, barbiturate intravenousanesthetics, benzodiazepine intravenous anesthetics, and opiate agonistintravenous anesthetics; hematological agents, such as antianemiaagents, hematopoietic antianemic agents, coagulation agents,anticoagulants, hemostatic coagulation agents, platelet inhibitorcoagulation agents, thrombolytic enzyme coagulation agents, and plasmavolume expanders, hormones and hormone modifiers, such asabortifacients, adrenal agents, corticosteroid, adrenal agents,androgens, anti-androgens, antidiabetic agents, sulfonylureaantidiabetic agents, antihypoglycemic agents, oral contraceptives,progestin contraceptives, estrogens, fertility agents, oxytocics,parathyroid agents, pituitary hormones, progestins, antithyroid agents,thyroid hormones, and tocolytics; immunobiologic agents, such asimmunoglobulins, immunosuppressives, toxoids, and vaccines; localanesthetics, such as amide local anesthetics and ester localanesthetics; musculoskeletal agents, such as anti-gout anti-inflammatoryagents, corticosteroid anti-inflammatory agents, gold compoundanti-inflammatory agents, immunosuppressive anti-inflammatory agents,,nonsteroidal anti-inflammatory drugs, salicylate anti-inflammatoryagents, skeletal muscle relaxants, neuromuscular blocker skeletal musclerelaxants, and reverse neuromuscular blocker skeletal muscle relaxants;neurological agents, such as anticonvulsants, barbiturateanticonvulsants, benzodiazepine anticonvulsants, anti-migraine agents,anti-parkinsonian agents, anti-vertigo agents, opiate agonists, andopiate antagonists, ophthalmic agents, such as anti-glaucoma agents,anti-glaucoma agents, mitotics, anti-glaucoma, agents, mydriatics,adrenergic agonist mydriatics, autimuscarinic mydriatics, ophthalmicanesthetics, ophthalmic anti-infectives, ophthalmic aminoglycosideanti-infectives, ophthalmic macrolide anti-infectives, ophthalmicquinolone anti-infectives, ophthalmic sulfonamide anti-infectives,ophthalmic tetracycline anti-infectives, ophthalmic anti-inflammatoryagents, ophthalmic corticosteroid anti-inflammatory agents, andophthalmic nonsteroidal anti-inflammatory drugs; psychotropic agents,such as antidepressants, heterocyclic antidepressants, monoamine oxidaseinhibitors selective serotonin re-uptake inhibitors tricyclicantidepressants, antimanics, antipsychotics, phenothiazineantipsychotics, anxiolytics, sedatives, and hypnotics, barbituratesedatives and hypnotics, benzodiazepine anxiolytics, sedatives, andhypnotics, and psychostimulants; respiratory agents, such asantitussives, bronchodilators, adrenergic agonist bronchodilators,antimuscarinic bronchodilators, expectorants, mucolytic agents,respiratory anti-inflammatory agents, and respiratory corticosteroidanti-inflammatory agents; toxicology agents, such as antidotes, heavyagents, substance abuse agents, deterrent substance abuse agents, andwithdrawal substance abuse agents, minerals; and vitamins, such asvitamin A, vitamin vitamin C, vitamin D, vitamin E, and vitamin K.

Other classes of biologically active agents from the above categoriesinclude: (1) analgesics in general, such as lidocaine, other “caine”analgesics or derivatives thereof and nonsteroidal anti-inflammatorydrugs (NSAIDs) analgesics, including diclofenac, ibuprofen, ketoprofen,and naproxen; (2) opiate agonist analgesics, such as codeine, fentanyl,hydromorphone, and morphine; (3) salicylate analgesics, such as aspirin(ASA) (enteric coated ASA); (4) H1-blocker antihistamines, such asclemastine and terfenadine, (5) H2-blocker antihistamines, such ascimetidine, famotidine, nizadine, and ranitidine; (6) anti-infectiveagents, such as muopirocin; (7) antianaerobic anti-infectives, such aschloramphenicol and clindamycin; (8) antifungal, antibioticanti-infectives, such as amphotericin b, clotrimazole, fluconazole, andketoconazole; (9) macrolide antibiotic anti-infectives, such asazithromycin and erythromycin; (10) miscellaneous antibioticanti-infectives, such as and imipenem; penicillin antibioticanti-infectives, such as nafcillin, oxacillin, penicillin G, andpenicillin V; (12) quiuolone antibiotic anti-infectives, such asciprofloxacin and norfloxacin; (13) tetracycline antibioticanti-infectives, such as doxycycline, minocycline and tetracycline: (14)antituberculosis antimycobacterial anti-infectives such as isoniazid andrifampin; (15) antiprotozoal anti-infectives, such as atovaquone anddapsone; (16) antimalarial antiprotozoal anti-infectives, such aschloroquine and pyrimethamine; (17) anti-retroviral anti-infectives,such as ritonavir and zidovudine; (18) antiviral anti-infective agents,such as acyclovir, ganciclovir, interferon-α, and rimantadine; (19)alkylating antineoplastic agents, such as carboplatin and cisplatin;(20) nitrosourea alkylating antineoplastic agents, such as carmustine(BCNU); (21) antimetabolite antineoplastic agents, such as methotrexate;(22) pyrrolidine analog antineoplastic agents, such as fluorouravil(5-FU) and gemcitabine; (23) hormonal antineoplastics, such asgoserelin, leuprolide, and tamoxifen; (24) natural antineoplastics, suchas aldesleukin, interleukin-2, docetaxel, etoposide interferon α,paclitaxel, other taxane derivatives, and tretinoin (ATRA); (25)antibiotic natural antineoplastics, such as bleomycin, dactinomyciln,daunorubicin, doxorubicin, and mitomycin; (26) vinca alkaloid naturalantineoplastics, such as vinblastine and vincristine; (27) autonomicagents, such as nicotine; (28) anticholinergic autonomic agents, such asbenztropine and trihexyphenidyl; (29) antimuscarinic anticholinergicautonomic agents, such as atropine and oxybutynin; (30) ergot alkaloidautonomic agents, such as bromocriptine; (31) cholinergic agonistparasympathomimetics, such as pilocarpine, (32) cholinesterase inhibitorparasympathomimetics, such as pyridostigmine; (33) a-blockersympatholytics, such as prazosin; (34) D-blocker sympatholytics, such asatenolol; (35) adrenergic sympathomimetics, such as albuterol anddobutamine; (36) cardiovascular agents, such as aspirin (ASA) (entericcoated ASA); (37) D-blocker antianginals, such as atenolol andpropranolol; (38) calcium-channel blocker antianginals, such asnifedipine and verapamil; (39) nitrate antianginals, such as isosorbidedinitrate (ISDN); (40) cardiac glycoside antiarrhythmics, such as (41)class I antiarrhythmics, such as lidocaine, mexiletine, phenytoin,procainamide, and quinidine; (42) class antiarrhythmics, such asatenolol, metoprolol, propranolol, and timolol; (43) classantiarrhythmics, such as amiodarone; (44) class IV antiarrhythmics, suchas diltiazem and verapamil; (45) antihypertensives, such as prazosin;(46) angiotensin-converting enzyme inhibitor (ACE inhibitor)antihypertensives, such as captopril and enalapril; (47)antihypertensives, such as atenolol, metoprolol, nadolol, andpropanolol; (48) calcium-channel blocker antihypertensive agents, suchas dilitiazem and nifedipine; (49) central-acting adrenergicantihypertensives, such as clonidine and methyldopa, (50) diureticantihypertensive agents, such as amiloride, furosemide,hydrochlorothiazide (HCTZ), and spironolactone; (51) peripheralvasodilator antihypertensives, such as and minoxidil; (52) antilipemics,such as gemfibrozil and probucol; (53) bile acid sequestrantantilipemics, such as cholestyramine; (54) reductase inhibitorantilipemics, such as lovastatin and pravastatin; (55) inotropes, such,as amrinone, dobutamine, and dopamine; (56) cardiac glycoside inotropes,such as (57) thrombolytic agents, such as alteplase (TPA), anistreplase,streptokinase, and urokinase; (58) dermatological agents, such ascolchicine, isotretinoin, methotrexate, minoxidil, tretinoin (59)dermatological corticosteroid anti-inflammatory agents, such asbetamethasone and dexamethasone, (60) antifungal topicalanti-infectives, such as amphotericin clotrimazole, miconazole, andnystatin; (61) antiviral topical anti-infectives, such as acyclovir;(62) topical antineoplastics, such as (63) electrolytic and renalagents, such as lactulose; (64) loop diuretics, such as furosemide; (65)potassium-sparing diuretics, such as triamterene; (66) thiazidediuretics, such as hydrochlorothiazide (HCTZ), (67) uricosuric agents,such as probenecid; (68) enzymes such as and (69) thrombolytic enzymes,such as alteplase, antistreplase, streptokinase and urokinase; (70)antimetics, such as prochlorperazine; (71) salicylate gastrointestinalanti-inflammatory agents, such as sulfasalazine: (72) gastric acid-pumpinhibitor anti-ulcer agents, such as omeprazole; (73) 112-blockeranti-ulcer agents, such as cimetidine, famotidine, nizatidine,ranitidine; (74) digestants, such as pancrelipase; (75) prokineticagents, such as erythromycin; (76) opiate agonist intravenousanesthetics such as fentanyl; (77) hematopoietic antianemia agents, suchas (G-CSF), and (GM-CSF); (78) coagulation agents, such as factors 1-10(AHF 1-10); (79) anticoagulants, such as warfarin; (80) thrombolyticenzyme coagulation agents, such as alteplase, anistreplase,streptokinase and urokinase; (81) hormones and hormone modifiers, suchas bromocriptine; (82) abortifacients, such as methotrexate, (83)antidiabetic agents, such as insulin; (84) oral contraceptives, such asestrogen and progestin; (85) progestin contraceptives, such aslevonorgestrel and norgestrel; (86) estrogens such as conjugatedestrogens, diethlystilbestrol (DES), estrogen (estradiol, estrone, andestropipate); (87) fertility agents, such as clomiphene, human chorionicgonadotropin (HCG), and menotropins; (88) parathyroid agents such ascalcitonin, (89) pituitary hormones, such as desmopressin, goserelin,oxytocin, and vasopressin (ADH); (90) progestins, such asmedroxyprogesterone, norethindrone, and progesterone; (91) thyroidhormones, such as levothyroxine, (92) immunobiologic agents, such asinterferon beta-1b and interferon gamma-1b; (93) immunoglobulins, suchas immune globulin IM, IMIG, IGIM and immune globulin IVIG, IGIV; (94)amide local anesthetics, as lidocaine; (95) ester local anesthetics,such as benzocaine and procaine; (96) musculoskeletal corticosteroidanti-inflammatory agents, such as beclomethasone, betamethasone,cortisone, dexamethasone, hydrocortisone, and prednisone; (97)musculoskeletal anti-inflammatory immunosuppressives, such asazathioprine, cyclophosphamide, and methotrexate; (98) musculoskeletalnonsteroidal anti-inflammatory drugs such as diclofenac, ibuprofen,ketoprofen, ketorlac, and naproxen; (99) skeletal muscle relaxants, suchas and diazepam; (100) reverse neuromuscular blocker skeletal musclerelaxants, such as pyridostigmine; (101) neurological agents, such asnimodipine, riluzole, tacrine and ticlopidine; (102) anticonvulsants,such as carbarmazepine, gabapentin, lamotrigine, phenytoin, and valproicacid; (103) barbiturate anticonvulsants, such as phenobarbital andprimidone; (104) benzodiazepine anticonvulsants, such as clonazepam,diazepam, and lorazepam; (105) anti-agents, such as bromocriptine,levodopa, carbidopa, and pergolide; (106) anti-vertigo agents, such asmeclizine; (170) opiate agonists, such as codeine, fentanyilhydromorphone, methadone, and morphine; (108) opiate antagonists, suchas naloxone; (109) and glaucoma agents, such as timolol; (110) mitoticanti-glaucoma agents, such as pilocarpine; (111) ophthalmicaminoglycoside anti-infectives, such as gentamicin, neomycin, andtobramycin, (112) ophthalmic quinolone anti-infectives, such asciprofloxacin, norfloxacin, and ofloxacin; (113) ophthalmiccorticosteroid anti-agents, such as dexamethasone and prednisolone;(114) ophthalmic nonsteroidal anti-inflammatory drugs such asdiclofenac; (113) antipsychotics, such as clozapine, haloperidol, andrisperidone; (116) benzodiazepine anxiolytics, sedatives and hypnotics,such as clonazepam, diazepam, lorazepam, oxazepam, and prazepam; (117)psychostimulants, such as methylphenidate and pemoline; (118) such ascodeine; (119) bronchodilators, such as (120) adrenergic agonistbronchodilators, such as albuterol; (121) respiratory corticosteroidanti-inflammatory agents, such as dexamethasone; (122) antidotes, suchas flumazenil and naloxone; (123) heavy metal agents, such aspenicillamine; (124) deterrent substance abuse agents, such asdisulfiram, naltrexone, and nicotine; (125) withdrawal substance abuseagents, such as bromocriptine; (126) minerals, such, as iron, calcium,and magnesium; (127) vitamin B compounds, such as cyanocobalamin(vitamin B12) and niacin (vitamin B3); (128) vitamin C compounds, suchas ascorbic acid; and (129) vitamin D such as calcitriol.

Further, recombinant or cell-derived proteins may be used, such as;recombinant beta-glucan; bovine immunoglobulin concentrate; bovinesuperoxide dismutase; formulation comprising fluorouracil, epinephrine,and bovine collagen; recombinant hirudin (r-Hir), HIV-1 immunogen;recombinant human growth hormone recombinant EPO (r-EPO); gene-activatedEPO (GA-EPO); recombinant human hemoglobin (r-Hb); recombinant humanmecasermin (r-IGF-1); recombinant interferon β; lenograstim (G-CSF);olanzapine, recombinant thyroid stimulating hormone (r-TSH); andtopotecan.

Still further the following listing of peptides, proteins, and otherlarge molecules may also be used, such as interleukins 1 through 18,including mutants and analogues; interferons a, y, and which may beuseful for cartilage regeneration, hormone releasing hormone (LHRH) andanalogues, gonadotropin releasing hormone transforming growth factor(TGF); fibroblast growth factor (FGF); tumor necrosis factor-a y and y);nerve growth factor (NGP); growth hormone releasing factor (GHRF),epidermal growth factor (EGF), connective tissue activated osteogenicfactors, fibroblast growth factor homologous factor (FGFHF); hepatocytegrowth factor (HGF); insulin growth factor (IGF); invasion inhibitingfactor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7);somatostatin; thymosin-a-y-giobulin; superoxide dismutase (SOD); andcomplement factors, and biologically active analogs, fragments, andderivatives of such factors, for example, growth factors.

Members of the transforming growth factor (TGF) supergene family, whichare multifunctional regulatory proteins, may be incorporated in apolymer matrix of the present invention. Members of the TGF supergenefamily include the beta transforming growth factors (for example, TGP-β1, TGF-β2, TGF-β3); bone morphogenetic proteins (for example, BMP-1,BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9); heparin-bindinggrowth factors (for example, fibroblast growth factor (FGF), epidermalgrowth factor (EGF), platelet-derived growth factor (PDGF), insulin-likegrowth factor (IGF)), (for example, Inhibin A, Inhibin B), growthdifferentiating factors (for example, GDF-1); and Activins (for example,Activin A, Activin B, Activin AB). Growth factors can be isolated fromnative or natural sources, such as from mammalian cells, or can beprepared synthetically, such as by recombinant DNA techniques or byvarious chemical processes. In addition, analogs, fragments, orderivatives of these factors can be used, provided that they exhibit atleast some of the biological activity of the native molecule. Forexample, analogs can be prepared by expression of genes altered bysite-specific mutagenesis or other genetic engineering techniques.

Various forms of the biologically active agents may be used. Theseinclude, without limitation, such forms as uncharged molecules,molecular complexes, salts, ethers, esters, amides, prodrug forms andthe like, which are biologically activated when implanted, injected orotherwise placed into a subject.

In certain embodiments, other materials may be incorporated into subjectcompositions in addition to one or more biologically active agents. Forexample, plasticizers and stabilizing agents known in the art may beincorporated in compositions of the present invention. In certainembodiments, additives such as plasticizers and stabilizing agents areselected for their biocompatibility or for the resulting physicalproperties of the reagents, the setting or gelling matrix or the set orgelled matrix.

A composition of this invention may further contain one or more adjuvantsubstances or the like. Such additional materials may affect thecharacteristics of the resulting composition. For example, fillers, suchas bovine serum albumin (BSA) or mouse serum albumin (MSA), may beassociated with the polymer composition. In certain embodiments, theamount of filler may range from about 0.1 to about 50% or more by weightof the composition. Incorporation of such fillers may affect thesustained release rate of any encapsulated substance. Other fillersknown to those of skill in the art, such as carbohydrates, sugars,starches, saccharides, celluloses and polysaccharides, including andsucrose, may be used in certain embodiments on the present invention.

Buffers, acids and bases may be incorporated in the compositions toadjust pH. Agents to increase the diffusion distance of agents releasedfrom the composition may also be included.

The charge, lipophilicity or by hydrophilicity of a subject compositionmay be modified by employing an additive. For example, surfactants maybe used to enhance miscibility of poorly miscible liquids. Examples ofsuitable surfactants include dextran, polysorbates and sodium laurylsulfate. In general, surfactants are used in low concentrations,generally less than about 5%.

The specific method used to formulate the novel formulations describedherein is not critical to the present invention and can be selected froma physiological buffer (Felgner et. al., U.S. Pat. No. 5,589,466(1996)).

Therapeutic formulations of the product may be prepared for storage aslyophilized formulations or aqueous solutions by mixing the producthaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, diluents, excipients or stabilizers typicallyemployed in the art, i.e., buffering agents, stabilizing agents,preservatives, isotonifiers, non-ionic detergents, antioxidants andother miscellaneous additives, see Remington's Pharmaceutical Sciences,16th ed., Osol, ed. (1980). Such additives are generally nontoxic to therecipients at the dosages and concentrations employed, hence, theexcipients, diluents, carriers and so on are pharmaceuticallyacceptable.

An “isolated” or “purified” polymer of interest is substantially free ofcontaminating proteins from the medium or tissue source from which thepolymer is obtained, or substantially free of chemical precursors orother chemicals or reactants in the medium or reaction mixture usedwhich contains components that are chemically synthesized. Thus, anisolated or purified imidated polymer is substantially free ofnon-imidated polymer material and includes preparations of less thanabout 30%, 20%, 25%, 20%, 10%, 5%, 4%, 3%, 2.5%. 2%, 1.5% or 1% or less,(by dry weight) of non-imidated biopolymer contaminants.

As used herein, the terms “stability” and “stable” in the contest of aliquid formulation comprising a biopolymer of interest that is resistantto thermal and chemical aggregation, degradation or fragmentation undergiven manufacture, preparation, transportation and storage conditions,such as, for one month, for two months, for three months, for fourmonths, for five months, for six months or more. The “stable”formulations of the invention retain biological activity equal to ormore than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% under given manufacture,preparation, transportation and storage conditions. The stability ofsaid preparation can be assessed by degrees of aggregation, degradationor fragmentation by methods known to those skilled in the art.

The term, “carrier,” refers to a diluent, adjuvant, excipient or vehiclewith which the therapeutic is administered. Such physiological carrierscan be sterile liquids, such, as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a suitablecarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions also can be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene glycol, water ethanol and the like.The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents.

The compositions can take the form of solutions, suspensions, emulsions,powders, sustained-release formulations, depots and the like. Examplesof suitable carriers are described in “Remington's PharmaceuticalSciences,” Martin. Such compositions will contain an effective amount ofthe biopolymer of interest, preferably in purified form, together with asuitable amount of carrier so as to provide the form for properadministration to the patient. As known in the art the formulation willbe constructed to suit the mode of administration.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. Buffers are preferably present at aconcentration ranging from about 2 mM to about 50 mM. Suitable bufferingagents for use with the instant invention include both organic andinorganic acids, and salts thereof, such as citrate buffers (e.g.,monosodium citrate-disodium citrate mixture, citric acid-trisodiumcitrate mixture, citric acid-monosodium citrate mixture etc.), succinatebuffers (e.g., succinic acid-monosodium succinate mixture, succinicacid-sodium hydroxide mixture, succinic acid-disodium succinate mixtureetc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture,tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxidemixture etc.), fumarate buffers (e.g., fumaric acid-monosodium fumaratemixture, fumaric acid-disodium fumarate mixture, monosodiumfumarate-disodium fumarate mixture etc.), gluconate buffers (e.g.,gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxidemixture, gluconic acid-potassium gluconate mixture etc.), oxalatebuffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodiumhydroxide mixture, oxalic acid-potassium oxalate mixture etc.), lactatebuffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodiumhydroxide mixture, lactic acid-potassium lactate mixture etc.) andacetate buffers (e.g., acetic acid-sodium acetate mixture, aceticacid-sodium hydroxide mixture etc.). Phosphate buffers, carbonatebuffers, histidine buffers, trimethylamine salts, such as Tris, HEPESand other such known buffers can be used.

Preservatives may be added to retard microbial growth, and may be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, m-cresol,octadecyldimethylbenzyl ammonium chloride, benzyaconium halides (e.g.,chloride, bromide and iodide), hexamethonium chloride, alkyl parabens,such as, methyl or propyl paraben, catechol, resorcinol, cyclohexanoland 3-pentanol.

Isotonicifiers are present to ensure physiological isotonicity of liquidcompositions of the instant invention and include polhydric sugaralcohols, preferably trihydric or higher sugar alcohols, such asglycerin, erythritol, arabitol, xylstol, sorbitol and mannitol.Polyhydric alcohols can be present in an amount of between about 0.1% toabout 25%, by weight, preferably 1% to 5% taking into account therelative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols,amino acids, such as arginine, lysine, glycine, glutamine, asparagine,histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamicacid, threonine etc.; organic sugars or sugar alcohols, such as lactose,trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol,ribitol myoinisitol, galactitol, glycerol and the like, includingcyclitols such as inositol; polyethylene glycol; amino acid polymers;sulfur containing reducing agents, such as urea, glutathione, thiocticacid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodiumthiosulfate; low molecular weight polypeptides (i.e., <10 residues);proteins, such as human serum, albumin, bovine serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone,saccharides, monosaccharides, such as xylose, mannose, fructose orglucose; disaccharides, such as lactose, maltose and sucrose;trisaccharides, such as raffinose; polysaccharides, such as, dextran andso on. Stabilizers can be present in the range from 0.1 to 10,000 w/wper part of biopolymer.

Additional miscellaneous excipients include bulking agents, (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine or vitamin E) and cosolvents.

As used herein, the term “surfactant” refers to organic substanceshaving amphipathic structures, namely, are composed of groups ofopposing solubility tendencies, typically an oil-soluble hydrocarbonchain and a water-soluble ionic group. Surfactants can be classified,depending on the charge of the surface-active moiety, into anionic,cationic and nonionic surfactants. Surfactants often are used aswetting, emulsifying, solubilizing and dispersing agents for variouspharmaceutical compositions and preparations of biological materials.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic agent, as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stresseswithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80 etc.), polyoxamers (184, 188etc.), Pluronic® polyols and polyoxyethylene sorbitan monoethers(TWEEN-20®, TWEEN-80® etc.). Non-ionic surfactants may be present in arange of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

As used herein, the term, “inorganic salt,” refers to any compound,containing no carbon, that results from replacement of part or all ofthe acid hydrogen or an acid by a metal or a group acting like a metal,and often is used as a tonicity adjusting compound in pharmaceuticalcompositions and preparations of biological materials. The most commoninorganic salts are NaCl, KCl, NaH₂PO₄ etc.

The present invention provides liquid formulations of a biopolymerhaving a pH ranging from about 5.0 to about 7.0, or about 5.5 to about6.5, or about 5.8 to about 6.2, or about 6.0, or about 6.0 to about 7.5,or about 6.5 to about 7.0.

The instant invention encompasses formulations, such as, liquidformulations having stability at temperatures found in a commercialrefrigerator and freezer found in the office of a physician orlaboratory, such as from about −20° C. to about 5° C., said stabilityassessed, for example, by microscopic analysis, for storage purposes,such as for about 60 days, for about 120 days, for about 180 days, forabout a year, for about 2 years or more. The liquid formulations of thepresent invention also exhibit stability, as assessed, for example, byparticle analysis, at room temperatures, for at least a few hours, suchas one hour, two hours or about three hours prior to use.

Examples of diluents include a phosphate buffered saline, buffer forbuffering against gastric acid in the bladder, such as citrate buffer(pH 7.4) containing sucrose, bicarbonate buffer (pH 7.4) alone, orbicarbonate buffer (pH 7.4) containing ascorbic acid, lactose, oraspartame. Examples of carriers include proteins, e.g., as found in skimmilk, sugars, e.g., sucrose, or polyvinylpyrrolidone. Typically thesecarriers would be used at a concentration of about 0.1-90% (w/v) butpreferably at a range of 1-10% (w/v).

The formulations to be used for in vivo administration must be sterile.That can be accomplished, for example, by filtration through sterilefiltration membranes. For example, the formulations of the presentinvention may be sterilized by filtration.

The biopolymer composition will be formulated, dosed and administered ina manner consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the biopolymer to be administeredwill be governed by such considerations, and can be the minimum amountnecessary to prevent, ameliorate or treat a disorder of interest. Asused herein, the term “effective amount” is an equivalent phrase refersto the amount of a therapy (e.g., a prophylactic or therapeutic agent),which is sufficient to reduce the severity and/or duration of a disease,ameliorate one or more symptoms thereof, prevent the advancement of adisease or cause regression of a disease, or which is sufficient toresult in the prevention of the development, recurrence, onset, orprogression of a disease or one or more symptoms thereof, or enhance orimprove the prophylactic and/or therapeutic effect(s) of another therapy(e.g., another therapeutic agent) useful for treating a disease. Forexample, a treatment of interest can increase the use of a joint in ahost, based on baseline of the injured or diseases joint, by at least5%, preferably at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 35%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 100%.In another embodiment, an effective amount of a therapeutic or aprophylactic agent of interest reduces the symptoms of a disease, suchas a symptom of arthritis by at least 5%, preferably at least 10%, atleast 15%,, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 100%. Also used herein as anequivalent is the term, “therapeutically effective amount.”

Where necessary, the composition may also include a solubilizing agentand a local anesthetic such as lidocaine or other “caine” anesthetic toease pain at the site of the injection.

Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water-free concentrate in a sealed container, such as an ampule orsachet indicating the quantity of active agent. Where the composition isto be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampule of sterile waterfor injection or saline can be provided, for example, in a kit, so thatthe ingredients may be mixed prior to administration.

An article of manufacture containing materials useful for the treatmentof the disorders described above is provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for preventing ortreating, for example, a wound or a joint disease and may have a sterileaccess port (for example, the container may be a vial having a stopperpierceable by a hypodermic injection needle). The label on or associatedwith the container indicates that the composition is used for treatingthe condition of choice. The article of manufacture may further comprisea second container comprising a pharmaceutically acceptable buffer, suchas phosphate-buffered saline, Ringer's solution and dextrose solution.It may further include other materials desirable from a commercial anduser standpoint, including buffers, diluents, filters, needles, syringesand package inserts with instructions for use.

Biologically active agents and other additives may be incorporated intothe cross-linked synthetic polymer composition by admixture or added toa reagent preparation. Alternatively, the agents may be incorporatedinto the cross-linked polymer matrix by binding these agents to thefunctional groups on the polymers of interest. Such compositions mayinclude linkages that can be easily biodegraded, for example as a resultof enzymatic degradation, resulting in the release of the active agentor additive into the target tissue, where it will exert its desiredtherapeutic effect.

A simple method for incorporating biologically active agents containingnucleophilic groups into the cross-linked polymer composition, involvesmixing the active agent with a polyelectrophilic component prior toaddition of the polynucleophilic Component. By varying the relativemolar amounts of the different components of the reactive composition,it is possible to alter the net charge of the resulting cross-linkedpolymer composition, in order to prepare a matrix for the delivery of acharged compound such as a protein or ionizable drug. As such, thedelivery of charged proteins or drugs, which would normally diffuserapidly out of a neutral carrier matrix, can be controlled.

For example, if a molar excess of a component that is polynucleophilicis used, the resulting matrix may have a net positive charge and can beused to ionically bind and deliver negatively charged compounds.Examples of negatively charged compounds that can be delivered frontthese matrices include various drugs, cells, proteins, andpolysaccharides.

If a molar excess of a component that is polyelectrophilic is used, theresulting matrix has a net negative charge and can be used to ionicallybind and deliver positively charged compounds. Examples of positivelycharged compounds that can be delivered from these matrices includevarious drugs, cells, proteins, and polysaccharides.

The cross-linked polymer matrix compositions of the present inventioncan also be used to deliver various types of living cells or genes to adesired site of administration to form new tissue. The term “genes” asused herein is intended to encompass genetic material from naturalsources, synthetic nucleic acids, DNA, antisense DNA and RNA.

For example, mesenchymal stem cells can be delivered using polymermatrices to produce cells of the same type as the tissue into which theyare delivered. Mesenchymal stem cells may not differentiated andtherefore may differentiate to form various types of new cells due tothe presence of an active agent or the effects (chemical, physical,etc.) of the local tissue environment. Examples of mesenchymal stemcells include osteoblasts, chondrocytes and fibroblasts. For example,osteoblasts can be delivered to the site of a bone detect to produce newbone; chondrocytes can be delivered to the site of a cartilage defect toproduce new cartilage; fibroblasts can be delivered to produce collagenwherever new connective tissue is needed; neurectodermal cells can bedelivered to form new nerve tissue; epithelial cells can be delivered toform new epithelial tissues, such as liver, pancreas etc.

The cells may be either allogeneic or xenogeneic in origin. Thecompositions can be used to deliver cells of species that aregenetically modified.

In some embodiments, the compositions of the invention may not easily bedegraded in vivo. Thus, cells entrapped within the cross-linked polymermatrix compositions will be isolated from the host cells and, as such,will not provoke or will delay an immune response in the host.

To entrap the cells or genes within a cross-linked polymer matrix, thecells or genes may, for example be pre-mixed with a reagent compositionor optionally with a mixture prior to forming a cross-linked polymermatrix, thereby entrapping the cells or genes within the matrix.

In a general method for effecting treating of an articulating surface ora disk or a joint, in the spine and so on within the body of a mammaliansubject, the components of the reactive composition are infused to thesite in need of treatment. The present invention may be prepared toinclude an appropriate vehicle for this injection, implantation,infusion or direction. Once at the body site of interest the imidatedbiologically compatible polymer reacts with and becomes fixed to asurface, such as a tissue or a prosthesis. Thus, the imidated polymer is“biologically anchored” to the host tissue and can then react with othersurfaces or polymers, such as the bridging molecule of interest.

The polymer matrix, alternatively, may be formed semi-solid or as asolid object implantable in an anatomic area, or as a film or mesh thatmay be used to cover a segment of an area or surface. A variety oftechniques for implanting solid objects in relevant anatomic areas willbe likewise familiar to the artisan.

In some embodiments, compositions disclosed herein may be positioned ina surgically created defect that is to be reconstructed, and is to beleft in that position after the reconstruction has been completed. Thepresent invention may be suitable for use with local tissuereconstructions, pedicle flap reconstructions, corneal flap sealings orfree flap reconstructions.

In some embodiments, this invention is directed to kits for bringingtissues or tissue parts into proximity, such as for sealing or healing.

The kits disclosed herein will include a container means for an imidatedpolymer of interest. The kit may include a delivery device. The kitoptionally will include a container means for a second polymer ofinterest. Instructions for their use can be included.

Uses for such kits include, for example, therapeutic applications. Theinvention provides kits for use in treating a disease or condition. Forexample, the kit may comprise an imidated biologically compatiblepolymer, such as, imidated chondroitin sulfate, and a biocompatiblepolymer or a compound comprising an amine moiety, such as, PEG-amine.

In certain embodiments, a polymer of interest can be formed into desiredstructures, such as films, foams, scaffolds or other three-dimensionalstructures of interest. In such circumstances, other materials may beincorporated into subject compositions, in addition to one or morebiologically active agents. For example, plasticizers and stabilizingagents known in the art may be incorporated in compositions of thepresent invention. The solid structure can be a component of a kit.Thus, an imidated biologically compatible polymer of interest may beapplied to a biological surface as a solid structure and enabled toreact with the biological surface. The bridging molecule then can bebrought into proximity with the affixed biologically compatible polymerto react therewith.

In other embodiments, the biologically compatible polymer is usedwithout the bridging molecule. Thus, the imidated biologicallycompatible polymer is used as an adhesive. The polymer can be applied inliquid form to a biological surface of interest. Alternatively, thepolymer can be combined with an inert structure, which can providesupport or serve as a carrier for the polymer, such as a backing for anadhesive bandage, or with a structure or device having a desiredfunction.

The imidated polymer of interest can be exposed to a first and a secondtissue and allowed to react therewith simultaneously. The imide groupsreact with functional groups on a tissue, such as the amino groups ofprotein lysines.

In another embodiment, the imide or one of the imides or the otherfunctional group is reactive not with a tissue but with anothersubstance for use in a body, such as a prosthesis, a hydrogel, ascaffold, a matrix and so on. Thus, the functionalized polymer ofinterest can be used to secure that substance to a tissue or to aparticular site in a body.

The compositions disclosed herein may be used in any number of tissuerepair applications, such as, but not limited to, seroma and hematomaprevention, skin and muscle flap attachment, repair and prevention ofendoleaks, aortic dissection repair, lung volume reduction, neural tuberepair, sealing of corneal incisions, reattaching a retina and themaking of microvascular and neural anastomoses.

In one embodiment, the repair of damaged tissue may be carried outwithin the context of any standard surgical process allowing access toand repair of the tissue, including open surgery and laparoscopictechniques. Once the damaged tissue is accessed, a composition of theinvention, is placed in contact with the damaged tissue along with anysurgically acceptable patch or implant, if needed. When used to repairlacerated or separated tissue, such, as by joining two or more tissuesurfaces, for example, following a surgical procedure, the compositionmay be applied to one or more of the tissue surfaces and then thesurfaces are placed in contact with each other and adhesion occurstherebetween.

When used to repair herniated tissue, a surgically acceptable patch canbe attached to the area of tissue surrounding the herniated tissue so asto cover the herniated area, thereby reinforcing the damaged tissue andrepairing the detect. When attaching the patch to the surroundingtissue, a composition of the invention may be applied to either thepatch, to the surrounding tissue, or to the patch after the patch hasbeen placed on the herniated tissue. Once the patch and tissue arebrought into contact with each other, adhesion may occur therebetween.

The surfaces to be adhered may be held together manually, or using otherappropriate means, such as adhesive tape, a temporary suture and so on,while the cross-linking reaction is proceeding to completion.Cross-linking is typically sufficiently complete tor adhesion to occurwithin about 5 to 60 seconds after mixing the components of the adhesivecomposition unless delayed setting is desired. However, the timerequired for complete cross-linking to occur is dependent on a number offactors, including the type and molecular weight of each reactivecomponent, the degree of functionalization, and the concentration of thecomponents in the cross-linkable compositions (e.g., higher componentconcentrations result in faster cross-linking times).

In one embodiment the compositions of the present invention aredelivered to the site of administration using an apparatus that allowsthe components to be delivered separately, for example, sequentially orsimultaneously. Such delivery systems may involve a multi-compartmentspray device.

Alternatively, the components can be delivered separately using any typeof controllable extrusion system, or they can be delivered manually inthe form of separate pastes, liquids or dry powders, and mixed togethermanually at the site of administration. Many devices that are adaptedfor delivery of multi-component tissue sealants/hemostatic agents arewell known in the art and can also be used in the practice of thepresent invention.

Yet another way of delivering the compositions of the present inventionis to prepare the reactive components in inactive form as either aliquid or powder. Such compositions can then be activated afterapplication to the tissue site, or immediately beforehand, by hydratingor applying an activator, for example. In one embodiment, the activatoris a buffer solution, having a pH that will activate the compositiononce mixed therewith. Still another way of delivering the compositionsis to prepare preformed sheets, and apply the sheets as such to the siteof administration. One of skill in the art can easily determine theappropriate adminstration protocol to use with any particularcomposition having a known gel strength and gelation time.

The compositions described herein can be used for medical conditionsthat require a coating or sealing layer to prevent the leakage of gases,liquid or solids. The method entails applying reagent(s) to the damagedtissue or organ to seal 1) vascular and/or other tissues or organs tostop or minimize the flow of blood; 2) thoracic tissue to stop orminimize the leakage of air; 3) gastrointestinal tract or pancreatictissue to stop or minimize the leakage of fecal or tissue contents; 4)bladder or ureters to stop or minimize the leakage of urine; 5) dura tostop or minimize the leakage of CSF; and 6) skin or serosal tissue tostop the leakage of serosal fluid. These compositions may also be usedto adhere tissues together such as small vessels, nerves or dermaltissue. The material can be used 1) by applying it to the surface of onetissue and then a second tissue may be rapidly pressed against the firsttissue or 2) by bringing the tissues in close juxtaposition and thenapplying the material. In addition, the compositions can be used to fillspaces in soft and hard tissues that are created by disease or surgery.

For example, polymer matrix compositions of the invention can be used toblock or fill various lumens and voids in the body of a mammaliansubject. The compositions can also be used as biosealants to sealfissures or crevices within a tissue or structure (such, as a vessel),or junctures between adjacent tissues or structures, to prevent leakageof blood or other biological fluids.

The compositions can also be used as a large space-filling device fororgan displacement in a body cavity during surgical or radiationprocedures, for example, to protect the intestines during a plannedcourse of radiation to the pelvis.

The compositions of the invention can also be coated onto the interiorsurface of a physiological lumen, such as a blood vessel or Fallopiantube, thereby serving as a sealant to prevent restenosis of the lumenfollowing medical treatment, such as, for example, ballooncatheterization to remove arterial plaque deposits from the interiorsurface of a blood vessel, or removal of scar tissue or endometrialtissue from the interior of a fallopian tube. A thin layer of thereaction mixture is preferably applied to the interior surface of thevessel (for example, via catheter) immediately following mixing of thefirst and second synthetic polymers. Because the compositions of theinvention are not readily degradable in vivo, the potential forrestenosis due to degradation of the coating is minimized.

The compositions of the invention can also be used for augmentation ofsoft or hard tissue within the body of a mammalian subject. Examples ofsoft tissue augmentation applications include sphincter (e. g., urinary,anal, esophageal) augmentation and the treatment of rhytids and scars.Examples of hard tissue augmentation applications include the repairand/or replacement of bone and/or cartilaginous tissue.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLE 1

Donor corneoscleral rims not suitable for transplantation are obtained.Corneas are preserved under standard eye bank conditions in Optisol-GSmedium (Bausch & Lomb Surgical, Inc, San Diego, Calif.) at 4° C. Theprocedure is performed no longer than 10 days after death, as taught inReyes et al., Invest. Ophthal. Vis. Sci. 46(4)1247 (2005), who reportedon the use of an aldehyde derivatized chondroitin sulfate withpolyvinylalcohol to seal corneal incisions. However, the toxicity ofthose reagents was not determined.

A manual microkeratome (LSK One; Moria USA, Doylestown, Pa.) is used toperform a hinged-flap keratectomy just past the central opening of thechamber, in a way that a large hinge is obtained. This opening issimilar to an artificial non-dilated pupil, which could be the referencepoint in a clinical setting. A 300 μm head thickness is used in allcorneas. An artificial anterior chamber (ALTK System; Moria USA) is usedto support the corneoscleral rims, as known in the art. The gearlesstracks on the base plate of the artificial anterior chamber are designedto fit into the microkeratome head, so that in its pass across thecornea it maintains the same plane and direction. All discs withposterior stroma, Descemet's membrane and endothelial cell layer areobtained using a 6.25 mm freehand trephine.

Infusion of isotonic sodium chloride is released before thecorneoscleral rims are placed on the base of the anterior chamber toclear the residual air from both the infusion line and underneath thecornea. The solution bottle is raised 1.5 m above the level of thechamber to obtain adequate intrachamber pressures (60-70 mm Hg) for themicrokeratome pass. Corneas are centered according to circular guides inthe base of the chamber. Mechanical epithelial scraping is performedwith a 2.5 mm straight, rounded tip crescent knife (Beaver, BectonDickinson Surgical Systems, Franklin Lakes, N.J.) to avoid surfaceirregularities due to loose epithelium, which may introduce errors inpachymetric and videokeratographic measurements.

The artificial anterior chamber is set to achieve a maximal flapdiameter in all cases. The maneuver is intended to leave as much area inthe stromal bed as possible for performing the trephination and suturingof the flap. The surgeries are all performed by the same surgeon toavoid variability related to different surgeons, using a surgicalmicroscope (Ophthamic 900S; Moeller-Wedel, Hamburg, Germany).

Several drops of proparacine hydrochloride are applied to the cornealsurface prior to the microkeratome pass to resemble clinical conditions.A partial flap keratectomy is performed by passing the microkeratomehead with its oscillating blade at a relatively constant speed acrossthe plate stepping just past the central opening of the chamber. Thisapproach differs from previously published techniques, in an attempt toobtain a wide flap binge with a relatively less likelihood of flapslippage, so that more stability to the corneal flap is added and thecorneal opening is reduced. The remaining stroma underneath the flapbinge is severed using a 2 mm wide Culler iris spatula (Sparta SurgicalCorporation, Concord, Calif.), as to leave adequate space to perform acentral trephination. Intrachamber pressure is returned to 18 to 20 mmHgby lowering the height of the isotonic sodium chloride solution bottleto 25 cm above the cornea level, and the trephine is centered accordingto the keratectomy and “pupillary” edge provided artificially by thecentral opening of the chamber. A hand trephine of 6.25 mm in diameteris used to perform a circular cut of the stromal bed. The trephine bladeis carefully rotated until perforation, and the remaining circular cutis completed with corneal scissors. Donor buttons are placed in therecipient beds, left unsutured, and the flap repositioned.

The experiment consists of two groups of four corneas each. In one group(Group 1), the flap is secured with five interrupted sutures (10-0Nylon, Sharpoint Surgical Specialties Corporation, Reading, Pa.). Thesuturing technique is the same in all corneas to ensure consistency.

In the second group (Group 2), the flap is secured using a tissueadhesive based on imidated chondroitin sulfate and PEG amine.

The bridging component, 10% PEG-amine, is used to overlay the CS-I. ThePEG also is intentionally stained blue with a biocompatible dye(Cibacron Blue; Sigma-Aldrich). Staining the bridging component permitsdirect observation of the polymerized glue relative to the incision andensures that the glue does not gain entry into the anterior chamber.

A 2.5-mm, straight, rounded-tip, crescent knife (Beaver; BD SurgicalSystems) is used to apply the CS-imide to the wound margins. A thinlayer is used to coat the surface of the incision and the internal woundlip. With a second crescent knife, a thin layer of bridging component ofthe adhesive (PEG-amine) is then applied over the first layer. The twocomponents are allowed to polymerize for 30 seconds. Once the gluesolidifies, saline is infused.

In both groups, the transplanted disc is left without sutures or glue,as it tends to keep in place by surface tension after the intrachamberpressure reaches 15-18 mmHg.

After epithelium removal, the isotonic sodium chloride infusion isclosed, and corneal thickness is measured using an ultrasound pachymeter(Pach IV, Accutome Inc, Malvern, Pa.) in the center of the cornea. Asecond measurement is made alter the hinged flap is created andreflected from the stromal bed. Central flap thickness is thencalculated.

For surface curvature analysis, a commercial videokeratoscope (EyeSysLaboratories, Inc, Houston, Tex.) is used. The Placido disc is placed ina vertical position and the chamber centered according to the monitorcontrol. Care is taken to preserve the orientation in preoperative andpostoperative recordings. Three measurements are performed preoperativeand postoperatively for each cornea.

To assess graft stability, intrachamber pressure is raisedprogressively. Presence of leakage is monitored and pressure is recordedby a digital manometer (Digimano 1000, Netech Corp, Hicksville, N.Y.).

Calculations are made using StatsDirect, version 1.9.0, for Windows(CamCode, Ashwell, England). Mean, SD, minimum and maximum values aredescribed.

The tissue adhesive produces good sealing and less astigmatism thanother reports of microkeratome-assisted posterior lamellar keratoplasty.Furthermore, the absence of sutures makes the technique simple andconsiderably less time consuming.

The composition of the instant invention can be used to adhere twoseparated surfaces, at least one of which is a biological surface. Thus,the instant composition can be used to seal a wound or an opening bybringing the open edges together in juxtaposition. The sealing can belong term or can be short term based on the level of biodegradability ofthe components of the instant adhesive. A short term seal can provide asuitable time for a healing or a natural sealing of the opening tooccur. Alternatively, the adhesive of interest can be used to adhere anon-biological but biocompatible surface to a biological surface. Such anon-biological surface can be found, for example, on a prosthesis, amedical device and so on.

EXAMPLE 2

The method of CS-NHS synthesis using carbodiimide was as known in theart. The imide derivative significantly improved efficacy andbiocompatibility. A CS-amine to act as the amine donor also wassynthesized. For example, an about 3:3:1 ratio of CS, succinimide anddiimide, respectively can be reacted in a small volume of saline for ashort period of time. A suitable ratio of the three reagents can beabout 75:100:38, as a design choice.

In another embodiment, CS (750 mg) was dissolved in 6 ml, PBS (phosphatebuffered saline), 1-Ethyl-3-[3-dimethylamino-propyl]carbodiimide (EDC,1.572 g, 8.2 mmol) was dissolved in 1.5 mL PBS. A 3.3 mmol solution ofN-hydroxysuccinimide (NHS) was made by dissolving 380 mg in 1.5 mL PBS.The NHS solution and the EDC were added to the CS solution, vortexed,and allowed to react for 10 minutes at 37° C. The reaction was thenchilled for 30 min at −80° C. and precipitated with ethanol. Thesolution was then centrifuged for 5 min and the supernatant was removedand washed.

Crosslinked CS networks were synthesized with varying ratios of NHS:NH₂as listed in the Table below. Polymer solutions with a concentration of10% (w/v) were made with 1:1, 1:2 and 2:1 ratios of CS-NHS toPEG-(NH₂)₆. PEG-(NH₂)₆ and CS-NHS were dissolved in DMEM to yield 3different concentrations; 13.3%, 10% and 6.67% (w/v). CS-NHS (50 μL) wasadded to a mold followed by the addition of 50 μL PEG-(NH2)6 and mixing.After 10 minutes, the networks were removed from the molds andtransferred to PBS for swelling ratio measurements.

50 μL CS-NHS 50 μL PEG-amine Description (% w/v) (% w/v) 1:1CS-NHS:PEG-amine 10 10 1:2 CS-NHS:PEG-amine 6.67 13.3 2:1CS-NHS:PEG-amine 13.3 6.67

The crosslinked networks were then evaluated with respect to swellingand cytocompatibility (cells encapsulated in the networks or culturedadjacent). The swelling properties are critical to ocular adhesiveapplications since excess swelling can open the sealed wound or causestigmatism. The networks were created with and without encapsulatedcells. The potential delivery of cells within the adhesive hasapplicability for larger corneal wounds that require some new tissueformation in addition to sealing of the wound. The CS networks werecomparable to the PEG control networks in all reports.

Fibroblasts were encapsulated in a CS-PEG amine network at varyingratios, 1:1, 1:2 and 2:1. Control PEG networks were produced at aconcentration of 5%, 10% and 20% w/v. Cells were stained for viabilityusing a commercially available kit. The amount of live cells in all gelswas comparable, indicating the CS-I based gels were biocompatible.

In another experiment, the CS-NHS and armed PEG were dissolved in PBScarrying differing amounts of HEPES buffer, for example, 10 mM, 100 mM,500 mM and 1000 mM HEPES. The reagents were mixed until gelationoccurred and pipetting of the reagents was no longer possible. It wasnoted that gelation time plateaued at about 100 mM HEPES. A slightdecrease in gel volume was noted with increasing HEPES concentrationsuggesting increasing crosslinking with increasing HEPES concentration.Modulus, or gel stiffness, generally, also increased with increasingHEPES concentration. At 500 mM, the modulus retreated a small amount,with a greater standard error.

Cells from nucleus pulposus, annnlus fibrous, chondrocytes, keratocytes,cornea endothelial cells, cornea epithelia cells and mesenchymal stemcells were tested for cytotoxicity with various gels of the instantinvention. Cells also were encapsulated in various gels of interest.Cells were monitored for at least over a 21 day period. As a control,cells were exposed to 5% PEG diacrylate. Gels contained a 1:2, 1:1 or2:1 ratios of CS to PEG. Some gels contained hyaluronic acid (HA) orglucosamine (GlcN), generally a 1:1 CS to PEG gel containing an equalpart of HA or GlcN. In all circumstances, cell viability was maintainedover the 21 day testing period. Cornea endothelia cells on day 8presented with a level of cell proliferation, an anti-apoptotic effectwas observed.

In another set of experiments, collagen was added to the CS-PEG gelsconstructed as described above to a final concentration of about 0.15%(w/v) A 1.75±0.08 fold increase of modulus was observed as compared toCS-PEG gels without collagen. It was contemplated that the collagen actsas a particulate in the gel matrix and thus endows the gel withcomposite properties.

The primary amines of collagen may interact with the CS-NHS groups inthe gel of interest. Hence, collagen may crosslink with the gel matrixvia covalent bonding. Such an increase in crosslinking may lead to adecrease in adhesiveness of the gel, because of the decrease in thenumber of available CS-NHS groups. One approach to avoid covalentinteraction between collagen and the gel matrix is to use afunctionalized collagen, such as one in which the amine group issubstituted, to minimize the reactivity of the amine group. For example,the amine group can be modified to contain an acetyl group, an alkylgroup, and so on, as taught hereinabove. That would lead to a gel withincreased modulus without sacrificing tissue adhesiveness.

Contemplated equivalents of the polymers, polymeric matrices, subunitsand other compositions described herein include such materials whichotherwise correspond thereto, and which have the same general propertiesthereof wherein one or more simple variations of substituents are madewhich do not adversely affect the efficacy of such molecule orcomposition to achieve its intended purpose. In general, the compoundsof the present invention may be prepared by the methods illustrated inthe general reaction schemes as, for example, described above, or bymodifications thereof using readily available starting materials,reagents and conventional synthesis procedures. In these reactions, itis also possible to make use of variants which are in themselves known,but are not mentioned here.

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

1.-9. (canceled)
 10. A method for filling a defect in a tissue or organcomprising applying to a defect in a tissue or organ a compositioncomprising a hydrophilic biologically compatible first polymer, whereinthe first polymer is isolated and purified chondroitin sulfatefunctionalized with an imide group, and at least one second hydrophilicbiologically compatible polymer, wherein said second polymer comprisesat least one amine group and is capable of reacting with the firstpolymer, the second polymer being selected from the group consisting ofpoly(ethylene oxide), partially or fully hydrolyzed poly(vinyl alcohol),poly(propylene oxide) block copolymers, polyvinylpyrrolidone),poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propylene oxide)block copolymers, poloxamines, carboxymethyl cellulose, hydroxyalkylatedcellulose, polypeptides, polysaccharides, carbohydrates, polysucrose,dextran, heparin sulphate, keratan sulfate, heparin, alginate, gelatin,collagen, albumin, ovalbumin, poly(phosphoesters), poly(lactides),poly(glycolides), poly(caprolactones), poly(amides), poly(urethanes),poly(esteramides), poly(orthoesters), poly(dioxanones), poly(acetals),poly(ketals), poly(carbonates), poly(orthocarbonates),poly(phosphazenes), poly(hydroxybutyrates), poly(hydroxyl valerates),poly(alkylene oxalates), poly(alkylene succinates), poly(malic acids),polyvinylalcoholpoly(hydroxycellulose), chitin, chitosan, andcopolymers, terpolymers or combinations or mixtures thereof; andallowing the polymers to polymerize.
 11. The method of claim 10, whereinsaid first and second polymers comprise at least ten monomeric units.12. The method of claim 10, wherein said imide group comprisessuccinimide.
 13. The method of claim 10, wherein allowing the polymersto polymerize comprises contacting the polymers with a polymerizationinitiator.
 14. The method of claim 13, wherein the polymerizationinitiator is selected from the group consisting of light,electromagnetic radiation, and thermal energy.
 15. A method for in situpolymerization of a biocompatible polymer in a subject comprising: a)administering to the site in the subject a composition comprising ahydrophilic biologically compatible first polymer, wherein the firstpolymer is isolated and purified chondroitin sulfate functionalized withan imide group, and at least one second hydrophilic biologicallycompatible polymer, wherein said second polymer comprises at least oneamine group and is capable of reacting with the first polymer, thesecond polymer being selected from the group consisting of poly(ethyleneoxide), partially or fully hydrolyzed poly(vinyl alcohol),poly(propylene oxide) block copolymers, poly(vinylpyrrolidone),poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propylene oxide)block copolymers, poloxamines, carboxymethyl cellulose, hydroxyalkylatedcellulose, polypeptides, polysaccharides, carbohydrates, polysucrose,dextran, heparin sulphate, keratan sulfate, heparin, alginate, gelatin,collagen, albumin, ovalbumin, poly(phosphoesters), poly(lactides),poly(glycolides), poly(caprolactones), poly(amides), poly(urethanes),poly(esteramides), poly(orthoesters), poly(dioxanones), poly(acetals),poly(ketals), poly(carbonates), poly(orthocarbonates),poly(phosphazenes), poly(hydroxybutyrates), poly(hydroxyl valerates),poly(alkylene oxalates), poly(alkylene succinates), poly(malic acids),polyvinylalcoholpoly(hydroxycellulose), chitin, chitosan, andcopolymers, terpolymers or combinations or mixtures thereof; and b)polymerizing the polymer in the subject.
 16. The method of claim 15,wherein the polymer is placed in contact with a surface of a prosthesisin the subject.
 17. The method of claim 15, wherein allowing the polymerto polymerize comprises contacting the polymer with a polymerizationinitiator.
 18. The method of claim 16, wherein allowing the polymer topolymerize comprises contacting the polymer with a polymerizationinitiator.
 19. The method of claim 17, wherein the polymerizationinitiator is selected from the group consisting of light,electromagnetic radiation, and thermal energy.
 20. The method of claim18, wherein the polymerization initiator is selected from the groupconsisting of light, electromagnetic radiation, and thermal energy. 21.A method for adhering a tissue surface in a subject comprising: a)applying to a first surface of a tissue a composition comprising ahydrophilic biologically compatible first polymer, wherein the firstpolymer is isolated and purified chondroitin sulfate functionalized withan imide group, and at least one second hydrophilic biologicallycompatible polymer; b) applying to the first surface of the tissue atleast one second hydrophilic biologically compatible polymer, whereinsaid second polymer comprises at least one amine group and is capable ofreacting with the first polymer, the second polymer being selected fromthe group consisting of poly(ethylene oxide), partially or fullyhydrolyzed poly(vinyl alcohol), poly(propylene oxide) block copolymers,poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethyleneoxide)-co-poly(propylene oxide) block copolymers, poloxamines,carboxymethyl cellulose, hydroxyalkylated cellulose, polypeptides,polysaccharides, carbohydrates, polysucrose, dextran, heparin sulphate,keratan sulfate, heparin, alginate, gelatin, collagen, albumin,ovalbumin, poly(phosphoesters), poly(lactides), poly(glycolides),poly(caprolactones), poly(amides), poly(urethanes), poly(esteramides),poly(orthoesters), poly(dioxanones), poly(acetals), poly(ketals),poly(carbonates), poly(orthocarbonates), poly(phosphazenes),poly(hydroxybutyrates), poly(hydroxyl valerates), poly(alkyleneoxalates), poly(alkylene succinates), poly(malic acids),polyvinylalcoholpoly(hydroxycellulose), chitin, chitosan, andcopolymers, terpolymers or combinations or mixtures thereof; c) placingthe first surface of the tissue in contact with a second surface andallowing the polymers to polymerize.
 22. The method of claim 20, whereinthe second surface is a biological surface.
 23. The method of claim 20,wherein the second surface is a non-biological biocompatible surface.24. The method of claim 23, wherein the non-biological biocompatiblesurface is the surface of a prosthesis in the subject.
 25. The method ofclaim 20, wherein allowing the polymer to polymerize comprisescontacting the polymer with a polymerization initiator.
 26. The methodof claim 25, wherein the polymerization initiator is selected from thegroup consisting of light, electromagnetic radiation, and thermalenergy.